阅读说明：本技术 聚乙二醇化的破伤风神经毒素和张力减退的治疗 (Pegylated tetanus neurotoxin and hypotonia treatment ) 是由 T·麦克林 P·斯姆克 L·诺伯里 P·克洛伊 R·孔杜伊特 A·萨塞 于 2019-07-30 设计创作，主要内容包括：本发明涉及一种组合物,所述组合物包含：第一聚乙二醇化的破伤风神经毒素(PEG-TeNT)和第二破伤风神经毒素(TeNT),所述PEG-TeNT包含与聚乙二醇(PEG)缀合的破伤风神经毒素(TeNT)。本发明还涉及各种PEG-TeNT。本发明还涉及一种使用所述组合物或各种PEG-TeNT治疗张力减退的方法和一种包含所述组合物或各种PEG-TeNT的试剂盒。在一个实施方案中,所述张力减退是阻塞性睡眠呼吸暂停。(The present invention relates to a composition comprising: a first pegylated tetanus neurotoxin (PEG-TeNT) and a second tetanus neurotoxin (TeNT), the PEG-TeNT comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG). The invention also relates to various PEG-TeNTs. The invention also relates to a method for treating hypotonia using the composition or various PEG-TeNTs and a kit comprising the composition or various PEG-TeNTs. In one embodiment, the hypotonia is obstructive sleep apnea.)

1. A tetanus neurotoxin (TeNT) or fragment thereof comprising one or more amino acid substitutions surface serine → cysteine relative to SEQ ID NO: 1.

2. The TeNT of claim 1, wherein the substituted serine comprises one or more of S81C, S120C, S144C, S248C, S335C, S428C, S600C, S963C, S1041C, S1155C, and S1187C, relative to SEQ ID NO 1.

3. The TeNT of claim 1 or 2, wherein the substituted cysteine is conjugated to polyethylene glycol (PEG).

4. The TeNT of claim 3, wherein the PEG is about 2kDa, 5kDa, about 10kDa, or about 20 kDa.

5. The TeNT of claim 3 or 4, wherein the PEG is linear or branched.

6. A TeNT or fragment thereof, comprising, relative to SEQ ID NO: 1: R1225K; R1225E; W1288A; W1288Y; W1288F; W1288L; R1225K and W1288A; R1225E and W1288A; R1225K and W1288Y; R1225E and W1288Y; R1225K and W1288F; R1225E and W1288F; R1225K and W1288L; R1225E and W1288L; r1225 del; w1288 del; or R1225del and W1288 del; E270A; Y374A; E270A and Y374; g270 del; y374 del; or a combination thereof, wherein the TeNT or fragment is inactive.

7. A composition, comprising: (i) a first pegylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) a second TeNT, wherein the first PEG-TeNT is not conjugated to the second TeNT.

8. The composition of claim 7, wherein the first PEG-TeNT comprises a TeNT according to any one of claims 3-5.

9. The composition of claim 7 or 8, wherein the second TeNT is a TeNT according to any one of claims 1-6.

10. A method for treating hypotonia, the method comprising administering to a subject: (i) a first pegylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) a second TeNT.

11. Use of a first pegylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG) in the manufacture of a medicament for treating hypotonia in a subject administered a second TeNT.

12. Use of a second TeNT in the manufacture of a medicament for treating hypotonia in a subject administered a first pegylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG).

13. Use of: (i) a first pegylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) a second TeNT.

14. The composition of any one of claims 7 to 9, the method of claim 10, or the use of any one of claims 11 to 13, wherein the first PEG-TeNT or the second TeNT comprises a pegylated TeNT Light Chain (LC), a pegylated TeNT Heavy Chain (HC), or a pegylated TeNT fragment c (c).

15. The composition, method or use according to any one of claims 7 to 14, wherein the first PEG-TeNT or the second TeNT is a PEG-TeNT-HC comprising a pegylated HC, or a PEG-TeNT-LC-c comprising a pegylated LC and a pegylated c.

16. The composition, method or use according to any one of claims 7-15, wherein the first PEG-TeNT is PEG-TeNT-HC and the second TeNT is PEG-TeNT-LC-c.

17. The composition, method, or use of any of claims 7-16, wherein the second TeNT comprises an inactivated TeNT.

18. The composition, method or use of claim 17, wherein the inactivated TeNT comprises, relative to SEQ ID NO: 1: R1225K; R1225E; W1228A; W1288Y; W1288F; W1288L; R1225K and W1288A; R1225E and W1288A; R1225K and W1288Y; R1225E and W1288Y; R1225K and W1288F; R1225E and W1288F; R1225K and W1288L; R1225E and W1288L; r1225 del; w1288 del; or R1225del and W1288 del; E270A; Y374A; E270A and Y374; g270 del; y374 del; or a combination thereof.

19. The method or use of any one of claims 9 to 18, wherein the subject is administered:

PEG-TeNT comprising pegylated c (PEG-TeNT-c) until efficacy is reduced; then the

A composition comprising PEG-TeNT-HC and PEG-TeNT-LC-c until efficacy is reduced; then the

PEG-TeNT comprising PEGylated LC and PEGylated HC (PEG-TeNT-LC-HC).

20. The method or use according to any one of claims 9 to 19, wherein the hypotonia is obstructive sleep apnea.

21. The composition of any one of claims 7 or 14 to 18, wherein the composition is a therapeutic composition.

22. A pegylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG).

23. The PEG-TeNT according to claim 22, wherein the TeNT Light Chain (LC) is pegylated, the TeNT Heavy Chain (HC) is pegylated, or the TeNT fragment c (c) is pegylated.

24. The PEG-TeNT according to claim 22 or 23, wherein LC is pegylated and HC is pegylated (PEG-TeNT-LC-HC), or LC is pegylated and c is pegylated (PEG-TeNT-LC-c).

25. The PEG-TeNT according to any one of claims 22 to 24, wherein PEG is conjugated to a lysine residue.

26. The PEG-TeNT according to any of claims 22 to 24, wherein PEG is conjugated to a cysteine residue, optionally wherein the cysteine residue is a surface serine → cysteine substitution relative to SEQ ID NO: 1.

27. The PEG-TeNT of any one of claims 22-26, wherein said PEG has a molecular weight of about 5kDa, about 10kDa, or about 20 kDa.

28. A method for treating hypotonia, the method comprising administering the PEG-TeNT of any one of claims 22-27 to a subject, wherein the PEG-TeNT is not conjugated to a second TeNT.

29. Use of the PEG-TeNT of any one of claims 22-27 in the manufacture of a medicament for the treatment of hypotonia, wherein the PEG-TeNT is not conjugated to a second TeNT.

30. The method of claim 28 or the use of claim 29, wherein a first PEG-TeNT comprising a pegylated HC (PEG-TeNT-HC) and a second PEG-TeNT comprising a pegylated LC and a pegylated c (PEG-TeNT-LC-c) are administered to the subject.

31. The method or use of any one of claims 28 to 30, wherein the subject is administered:

PEG-TeNT comprising pegylated c (PEG-TeNT-c); and/or

A first PEG-TeNT comprising a PEGylated HC (PEG-TeNT-HC) and a second PEG-TeNT comprising a PEGylated LC-c (PEG-TeNT-LC-c); and/or

PEG-TeNT comprising PEGylated HC and PEGylated LC (PEG-TeNT-LC-HC).

32. The method or use of any one of claims 28 to 31, wherein treating comprises administering to the subject:

administering a PEG-TeNT comprising pegylated c (PEG-TeNT-c) until efficacy is reduced; then the

PEG-TeNT-HC and PEG-TeNT-LC-c are administered until efficacy is reduced; then the

PEG-TeNT comprising PEGylated LC and PEGylated HC (PEG-TeNT-LC-HC) is administered.

33. The method or use of any one of claims 28-32, wherein treating further comprises administering inactivated TeNT.

34. The method or use of claim 33, wherein the inactivated TeNT comprises, relative to SEQ ID NO: 1: R1225K; R1225E; W1288A; W1288Y; W1288F; W1288L; R1225K and W1288A; R1225E and W1288A; R1225K and W1288Y; R1225E and W1288Y; R1225K and W1288F; R1225E and W1288F; R1225K and W1288L; R1225E and W1288L; r1225 del; w1288 del; or R1225del and W1288 del; E270A; Y374A; E270A and Y374; g270 del; y374 del; or a combination thereof.

35. The method or use according to any one of claims 28 to 34, wherein the hypotonia is obstructive sleep apnea.

36. A kit comprising the TeNT of any one of claims 1 to 6, the composition of any one of claims 7, 14 to 18, or 21, or the PEG-TeNT of any one of claims 22 to 27.

Technical Field

The present invention relates to a composition comprising: a first pegylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG) and a second TeNT. The invention also relates to a method of treating hypotonia using the composition.

Background

Tetanus neurotoxin (TeNT) is produced by Clostridium tetani (Clostridium tetani). TeNT acts on the spinal cord and blocks the release of gamma-aminobutyric acid (GABA) and glycine, which are inhibitory neurotransmitters, at spinal inhibitory interneurons. Thus, TeNT causes spastic paralysis. TeNT does not exist in multiple serotypes.

Treatment with the biological properties of TeNT or at least TeNT fragments has been proposed. However, long-term treatment with protein therapeutics often results in a targeted immune response. Since only one serotype of TeNT is known, TeNT-based treatments cannot select serotype switching to circumvent immunity. In addition, many populations have been vaccinated against TeNT, thereby precluding TeNT-based therapy.

US 2002/0197278 a1 discloses the use of a pegylated botulinum toxin for the treatment of an inappropriate muscle contraction disorder. US 2002/0197278 a1 also proposes the use of pegylated TeNT for the treatment of inappropriate muscle contraction disorders, such as migratory headache or strabismus. However, as noted above, TeNT causes muscle contraction, thereby precluding its use for treating inappropriate muscle contraction conditions, with or without pegylation.

Wan et al, Process Biochemistry (2017)52:183-191, disclosed the effect of pegylation on the anti-PEG immune response resulting from administration of pegylated proteins, but did not utilize its discovery for any treatment.

WO 2016/001762 a1 discloses the use of pegylated TeNT fragment c (c) for increasing muscle mass. When TeNT was digested by papain, a fragment C (50kDa) was generated, and this fragment C corresponds to 451 amino acids at the C-terminal end of the TeNT heavy chain. Fragment c retains the binding, internalization, and transsynaptic transport capabilities of undigested TeNT, but does not disrupt any neuronal process, and is therefore non-toxic.

There is a need for a TeNT-based treatment that avoids a pre-existing anti-TeNT immune response in a subject vaccinated with tetanus toxoid.

It will be appreciated that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art in australia or any other country.

Disclosure of Invention

The present inventors have recognized that the use of TeNT has not been fully realized due to the adaptive immune system, which generates an antibody response upon administration of a protein therapeutic, thereby reducing the efficacy of the protein therapeutic. As a result of vaccination, the adaptive immune response may be deliberate, as exhibited by many people who have been immunized against TeNT. Alternatively, the adaptive immune response may be unintentional, resulting from repeated exposure to the protein therapeutic.

The inventors of the present application have generated a family and treatment regimen of modified TeNT that addresses these issues. In particular, the present invention provides a family and a hierarchical treatment regimen of pegylated (PEG-TeNT) tents that each evade the immune system, wherein a different alternative PEG-TeNT is used for treatment when the efficacy of a previously administered PEG-TeNT decreases or when the immunological profile of the patient precludes the use of another PEG-TeNT.

In a first aspect, there is provided tetanus neurotoxin (TeNT) or fragment thereof comprising one or more amino acid substitutions surface serine → cysteine relative to SEQ ID NO: 1.

In one embodiment of the first aspect, the substituted cysteine is conjugated to polyethylene glycol (PEG).

A second aspect provides a TeNT or fragment thereof comprising, relative to SEQ ID No. 1: R1225K; R1225E; W1288A; W1288Y; W1288F; W1288L; R1225K and W1288A; R1225E and W1288A; R1225K and W1288Y; R1225E and W1288Y; R1225K and W1288F; R1225E and W1288F; R1225K and W1288L; R1225E and W1288L; r1225 del; w1288 del; or R1225del and W1288 del; E270A; Y374A; E270A and Y374; g270 del; y374 del; or a combination thereof, wherein the TeNT or fragment is inactive.

A third aspect provides a composition comprising: (i) a first pegylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) a second TeNT, wherein the first PEG-TeNT is not conjugated to the second TeNT.

A fourth aspect provides a method for treating hypotonia, the method comprising administering to a subject: (i) a first pegylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) a second TeNT.

The fourth aspect also provides a use of a first pegylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG) in the manufacture of a medicament for treating hypotonia in a subject administered a second TeNT.

The fourth aspect also provides for use of a second tetanus neurotoxin (TeNT) in the manufacture of a medicament for treating hypotonia in a subject receiving a first pegylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG).

The fourth aspect also provides the use of: (i) a first pegylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) a second TeNT.

The fourth aspect also provides a first pegylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG), for use in a method of treating hypotonia in a subject administered a second TeNT.

The fourth aspect also provides a second tetanus neurotoxin (TeNT) for use in a method of treating hypotonia in a subject administered a first pegylated tetanus neurotoxin (PEG-TeNT) comprising a tetanus neurotoxin (TeNT) conjugated to a polyethylene glycol (PEG).

The fourth aspect also provides: (i) a first pegylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) a second TeNT; the compositions are used in methods of treating hypotonia.

In an embodiment of the fourth aspect, a composition can comprise the first PEG-TeNT and the second TeNT.

The fourth aspect also provides a composition comprising: (i) a first pegylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) a second TeNT, for use in a method of treating hypotonia.

In one embodiment, the first PEG-TeNT or the second TeNT comprises a pegylated TeNT Light Chain (LC), a pegylated TeNT Heavy Chain (HC), and a pegylated TeNT Light Chain (LC), or a pegylated TeNT fragment c (c). In one embodiment, the first PEG-TeNT or the second TeNT comprises PEG-TeNT-LC-HC.

In another embodiment, the first PEG-TeNT or the second TeNT is a PEG-TeNT-HC comprising a pegylated HC. In this embodiment, the LC is not pegylated. In another embodiment, the first PEG-TeNT or the second TeNT is PEG-TeNT-LC-c comprising pegylated LC and pegylated c. In this embodiment, HN is not pegylated.

In another embodiment, the first PEG-TeNT is PEG-TeNT-HC and the second TeNT is PEG-TeNT-LC-c.

In one embodiment, the second TeNT comprises an inactivated TeNT. Relative to SEQ ID NO:1, the inactivated TeNT may comprise: R1225K; R1225E; W1288A; W1288Y; W1288F; W1288L; R1225K and W1288A; R1225E and W1288A; R1225K and W1288Y; R1225E and W1288Y; R1225K and W1288F; R1225E and W1288F; R1225K and W1288L; R1225E and W1288L; r1225 del; w1288 del; r1225del or W1288 del; E270A; Y374A; E270A and Y374A; g270 del; y374 del; or a combination thereof. In one embodiment, the second TeNT comprises an inactivated TeNT comprising R1225E and W1288A.

In one embodiment, the subject is administered: PEG-TeNT comprising pegylated c (PEG-TeNT-c) until efficacy is reduced; then administering a composition comprising PEG-TeNT-HC and PEG-TeNT-LC-c until efficacy is reduced; PEG-TeNT (PEG-TeNT-LC-HC) containing PEGylated LC and PEGylated HC was then administered.

In one embodiment, treating comprises administering to the subject: PEG-TeNT comprising pegylated c (PEG-TeNT-c); then administering a composition comprising PEG-TeNT-HC and PEG-TeNT-LC-c; PEG-TeNT (PEG-TeNT-LC-HC) comprising pegylated LC and pegylated HC is then administered to determine an immunological profile of the subject's anti-TeNT antibodies and to determine an effective composition of PEG-TeNT based on the profile.

In one embodiment, the hypotonia is obstructive sleep apnea.

In another embodiment, the composition is a therapeutic composition. In another embodiment, the composition is a cosmetic composition.

Also disclosed is a method for producing a protein consisting of TeNT LC and TeNT HN (LC-HN), the method comprising expressing in a host cell a nucleic acid molecule encoding an amino acid sequence comprising SEQ ID No. 6.

A fifth aspect provides a pegylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG).

In one embodiment, the TeNT Light Chain (LC) is pegylated, the TeNT Heavy Chain (HC) is pegylated, or the TeNT fragment c (c) is pegylated. In one embodiment, the LC is pegylated and the HC is pegylated (PEG-TeNT-LC-HC), or the LC is pegylated and c is pegylated (PEG-TeNT-LC-c).

In another embodiment, PEG is conjugated to a lysine residue of TeNT. In another embodiment, PEG is conjugated to a cysteine residue of TeNT. In one embodiment, the PEG is conjugated to a cysteine residue of TeNT, wherein the cysteine residue is native or is a substitution for a serine residue, relative to SEQ ID NO: 1.

In one embodiment, the PEG has a molecular weight of about 2kDa, about 5kDa, about 10kDa, or about 20kDa, or about 30 kDa.

A sixth aspect provides a method for treating hypotonia, the method comprising administering the PEG-TeNT of the fifth aspect to a subject, wherein the PEG-TeNT is not conjugated to a second TeNT.

The sixth aspect also provides the use of the PEG-TeNT of the fifth aspect in the manufacture of a medicament for the treatment of hypotonia, wherein the PEG-TeNT is not conjugated to a second TeNT.

Alternatively, a sixth aspect provides the PEG-TeNT of the fifth aspect for use in a method of treating hypotonia, the method comprising administering the PEG-TeNT to a subject, wherein the PEG-TeNT is not conjugated to a second TeNT.

In one embodiment, a first PEG-TeNT comprising a pegylated HC (PEG-TeNT-HC) and a second PEG-TeNT comprising a pegylated LC and a pegylated c (PEG-TeNT-LC-c) are administered to the subject.

In another embodiment, the subject is administered: PEG-TeNT comprising pegylated c (PEG-TeNT-c); and/or a first PEG-TeNT comprising a pegylated HC (PEG-TeNT-HC) and a second PEG-TeNT comprising a pegylated LC-c (PEG-TeNT-LC-c); and/or PEG-TeNT comprising pegylated HC and pegylated LC (PEG-TeNT-LC-HC).

In one embodiment, treating comprises administering to the subject: PEG-TeNT comprising pegylated c (PEG-TeNT-c) until efficacy is reduced. Thereafter, treating may comprise administering to the subject: PEG-TeNT-HC and PEG-TeNT-LC-c until efficacy is reduced. Thereafter, treating may comprise administering to the subject: PEG-TeNT comprising PEGylated LC and PEGylated HC (PEG-TeNT-LC-HC).

In one embodiment, treating comprises administering to the subject: PEG-TeNT comprising pegylated c (PEG-TeNT-c) until efficacy is reduced; PEG-TeNT-HC and PEG-TeNT-LC-c are then administered until efficacy decreases; PEG-TeNT (PEG-TeNT-LC-HC) containing PEGylated LC and PEGylated HC was then administered.

In one embodiment, treating comprises administering to the subject: PEG-TeNT comprising pegylated c (PEG-TeNT-c) comprising PEG having a molecular weight of about 5kDa until efficacy decreases; then administering PEG-TeNT comprising pegylated c (PEG-TeNT-c) comprising PEG having a molecular weight of about 10kDa until efficacy decreases; PEG-TeNT comprising pegylated c (PEG-TeNT-c) comprising PEG with a molecular weight of about 20kDa is then administered until efficacy is reduced. Thereafter, treating may comprise administering to the subject: PEG-TeNT-HC and PEG-TeNT-LC-c, either or both of which comprise PEG having a molecular weight of 5kDa until efficacy decreases; then PEG-TeNT-HC and PEG-TeNT-LC-c, either or both of which comprise PEG having a molecular weight of 10kDa, are administered until efficacy is reduced; PEG-TeNT-HC and PEG-TeNT-LC-c, either or both of which comprise PEG having a molecular weight of 20kDa, are then administered until efficacy is reduced. Thereafter, treating may comprise administering to the subject: PEG-TeNT comprising pegylated LC and pegylated HC (PEG-TeNT-LC-HC), either or both of which comprise PEG having a molecular weight of 5kDa until efficacy is reduced; then administering PEG-TeNT comprising pegylated LC and pegylated HC (PEG-TeNT-LC-HC), either or both of which comprise PEG having a molecular weight of 10kDa, until efficacy decreases; PEG-TeNT (PEG-TeNT-LC-HC) comprising pegylated LC and pegylated HC, either or both of which comprise PEG having a molecular weight of 20kDa, is then administered.

In one embodiment, treating comprises administering to the subject: PEG-TeNT comprising pegylated c (PEG-TeNT-c); then administering a composition comprising PEG-TeNT-HC and PEG-TeNT-LC-c; PEG-TeNT (PEG-TeNT-LC-HC) comprising pegylated LC and pegylated HC is then administered to determine an immunological profile of the subject's anti-TeNT antibodies and to determine an effective composition of PEG-TeNT based on the profile.

In another embodiment, treatment with TeNT further comprises administration of inactivated TeNT. Relative to SEQ ID NO:1, the inactivated TeNT may comprise: R1225K; R1225E; W1228A; W1288Y; W1288F; W1288L; R1225K and W1288A; R1225E and W1288A; R1225K and W1288Y; R1225E and W1288Y; R1225K and W1288F; R1225E and W1288F; R1225K and W1288L; R1225E and W1288L; r1225 del; w1288 del; r1225del or W1288 del. In one embodiment, the second TeNT comprises an inactivated TeNT comprising R1225E and W1288A; E270A; Y374A; E270A and Y374A; g270 del; y374 del; or a combination thereof.

In one embodiment, the hypotonia is obstructive sleep apnea.

A seventh aspect provides a kit comprising the TeNT of the first aspect, the composition of the second aspect, or the PEG-TeNT of the fifth aspect.

In one embodiment, the composition or PEG-TeNT is used according to the method of the third or sixth aspect, respectively.

Drawings

FIG. 1 is a schematic of an exemplary PEG-TeNT of the present invention: a is PEG-TeNT-c; b is PEG-TeNT-HC; c is PEG-TeNT-LC-C; d is PEG-TeNT-LC-HC, wherein the TeNT is intact and active, i.e., the TeNT comprises two chains, and pegylation is present on a specific region, i.e., c, HC, LC-c or LC-HC, respectively.

FIG. 2 is the amino acid sequence of mature TeNT comprising 1314 amino acids (SEQ ID NO: 1).

FIG. 3 is the nucleic acid sequence of vector pRSET-TeNT encoding TeNT (SEQ ID NO: 2).

FIG. 4 is a diagram of vector pRSET-TeNT encoding TeNT. TeNT is expressed as having an N-terminal His₆And (4) a label. The nucleic acid was inserted into the MCS of the pRSET-A vector and mutexpressed under the control of the T7 promoter.

FIG. 5 is the amino acid sequence of HC (SEQ ID NO:3) comprising amino acids 457 to 1314 of SEQ ID NO: 1.

FIG. 6 is the amino acid sequence of c comprising amino acids 864 to 1314 of SEQ ID NO:1 (SEQ ID NO: 4).

FIG. 7 is the nucleic acid sequence of vector pRSET-TeNT-c encoding c (SEQ ID NO: 5).

FIG. 8 is a diagram of vector pRSET-TeNT-c encoding c. c is expressed as having an N-terminal His₆And (4) a label. The nucleic acid was inserted into the MCS of the pRSET-A vector and mutexpressed under the control of the T7 promoter.

FIG. 9 is the amino acid sequence of LC-HN comprising amino acids 1 to 863 of SEQ ID NO:1 (SEQ ID NO: 6).

FIG. 10 is the nucleic acid sequence of vector pRSET-TeNT-LC-HN encoding LC-HN (SEQ ID NO: 7).

FIG. 11 is a diagram of vector pRSET-TeNT-LC-HN encoding LC-HN. LC-HN is expressed as having an N-terminal His₆And (4) a label. The nucleic acid was inserted into the MCS of the pRSET-A vector and mutexpressed under the control of the T7 promoter.

FIG. 12 is the amino acid sequence of non-functional c comprising amino acid substitutions W1288A and R1225E relative to SEQ ID NO:1 (SEQ ID NO: 8).

FIG. 13 is the nucleic acid sequence (SEQ ID NO:9) of vector pRSET-TeNT-c encoding the non-functional c of FIG. 12 (SEQ ID NO:8) comprising the amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1.

FIG. 14 is a diagram of vector pRSET-TeNT-c encoding the non-functional c of FIG. 12 (SEQ ID NO:8) comprising amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1. C comprising the amino acid substitutions W1288A and R1225E relative to SEQ ID NO 1 was expressed as having an N-terminal His₆And (4) a label. The nucleic acid was inserted into the MCS of the pRSET-A vector and mutexpressed under the control of the T7 promoter.

FIG. 15 is the amino acid sequence of non-functional TeNT comprising the amino acid substitutions W1288A and R1225E relative to SEQ ID NO:1 (SEQ ID NO: 10).

FIG. 16 is the nucleic acid sequence (SEQ ID NO:11) of vector pRSET-TeNT encoding the non-functional TeNT of FIG. 15(SEQ ID NO:10) comprising the amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1.

FIG. 17 is a diagram of vector pRSET-TeNT encoding the non-functional TeNT of FIG. 15(SEQ ID NO:10) comprising the amino acid substitutions W1288A and R1225E relative to SEQ ID NO: 1. TeNT comprising the amino acid substitutions W1288A and R1225E relative to SEQ ID NO 1 was expressed with an N-terminal His₆And (4) a label. The nucleic acid was inserted into the MCS of the pRSET-A vector and mutexpressed under the control of the T7 promoter.

FIG. 18 is the amino acid sequence of mature TeNT having 1314 amino acids (SEQ ID NO:12) containing the amino acid substitutions S81C, S120C, S144C, S248C, S335C, S428C, S600C, S963C, S1041C, S1155C and S1187C of surface serine → cysteine relative to SEQ ID NO: 1.

FIG. 19 is the nucleic acid sequence (SEQ ID NO:13) of vector pRSET-TeNT encoding mature TeNT with cysteine substituted for surface serine of FIG. 18 (SEQ ID NO: 12).

FIG. 20 is a diagram of vector pRSET-TeNT of FIG. 19 (SEQ ID NO:13) encoding mature TeNT of FIG. 18 with surface serine substituted with cysteine (SEQ ID NO: 12). TeNT with surface serine substituted by cysteine was expressed as having N-terminal His₆And (4) a label. The nucleic acid was inserted into the MCS of the pRSET-A vector and mutexpressed under the control of the T7 promoter.

FIG. 21 is the amino acid sequence of mature TeNT having 1314 amino acids (SEQ ID NO:14) containing the amino acid substitutions S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C and S1187C of surface serine → cysteine in the LC region and the c region relative to SEQ ID NO: 1.

FIG. 22 is the nucleic acid sequence (SEQ ID NO:15) of vector pRSET-TeNT encoding mature TeNT (SEQ ID NO:14) with cysteine substituted for surface serine of FIG. 21.

FIG. 23 is a diagram of vector pRSET-TeNT of FIG. 22 (SEQ ID NO:15) encoding mature TeNT of FIG. 21 with surface serine substituted with cysteine (SEQ ID NO: 14). TeNT with surface serine substituted by cysteine was expressed as having N-terminal His₆And (4) a label. The nucleic acid was inserted into the MCS of the pRSET-A vector and mutexpressed under the control of the T7 promoter.

FIG. 24 is the amino acid sequence of TeNT comprising the HC surface serine → cysteine amino acid substitutions S600C, S963C, S1041C, S1155C and S1187C relative to SEQ ID NO:1 (SEQ ID NO: 16).

FIG. 25 is the nucleic acid sequence (SEQ ID NO:17) of vector pRSET-TeNT encoding mature TeNT with cysteine substituted for surface serine of FIG. 24 (SEQ ID NO: 16).

FIG. 26 is a diagram of vector pRSET-TeNT of FIG. 25 (SEQ ID NO:17) encoding TeNT having a surface serine substituted with cysteine (SEQ ID NO:16) of FIG. 24. TeNT with surface serine substituted by cysteine was expressed as having N-terminal His₆And (4) a label. The nucleic acid was inserted into the MCS of the pRSET-A vector and mutexpressed under the control of the T7 promoter.

FIG. 27 is a 3-dimensional protein structure model of TeNT derived from crystallographic data stored in the protein database (accession ID PDB: 5N0B) using Discovery Studio to map epitopes recognized by major human antibody clonotypes (as identified by da Silva antonnes et al (2017) and Palermo et al (2017)) onto the model. Surface serine residues in or near the identified epitope are selected to be mutated to cysteine for subsequent pegylation.

FIG. 28 includes two photographs of SDS-PAGE analysis of PEG-TeNT containing PEG with increasing molecular weight, where (A) detection was performed using Coomassie blue and (B) detection was performed by Western blotting using a polyclonal anti-TeNT antibody. (B) Immunogenicity is shown to be proportional to PEG molecular weight. The left border has a molecular weight marker in kDa, lane 1; TeNT lane 2; lane 3 of 2kDa PEG-TeNT-HC-LC; 5kDa PEG-TeNT-LC-HC lane 4; lane 5 10kDa PEG-TeNT-LC-HC; 20kDa PEG-TeNT-LC-HC lane 6.

Fig. 29 includes four line graphs representing competitive ELISA assays. Four PEG-TeNT and four PEG-TeNT-LC-c serine mutants each comprising PEG (2kDa, 5kDa, 10kDa and 20kDa) of different molecular weights were determined against TeNT using a polyclonal anti-TeNT antibody. (A) TeNT was adsorbed onto ELISA plates and then probed with polyclonal anti-TeNT antibodies pre-incubated with each of the four PEG-TeNT antigens (2kDa, 5kDa, 10kDa and 20kDa) at four concentrations (10. mu.g/mL, 1. mu.g/mL, 0.1. mu.g/mL and 0.01. mu.g/mL). (B) Each PEG-TeNT (2kDa, 5kDa, 10kDa and 20kDa) was adsorbed onto a separate ELISA plate and then probed with polyclonal anti-TeNT antibodies pre-incubated with each of the four concentrations (10. mu.g/mL, 1. mu.g/mL, 0.1. mu.g/mL and 0.01. mu.g/mL) of TeNT antigen. (C) The TeNT-LC-c serine mutant was adsorbed onto an ELISA plate and probed with polyclonal anti-TeNT antibodies pre-incubated with each of the four PEG-TeNT-LC-c serine mutant antigens (2kDa, 5kDa, 10kDa and 20kDa) at four concentrations (10. mu.g/mL, 1. mu.g/mL, 0.1. mu.g/mL and 0.01. mu.g/mL). (D) Each PEG-TeNT-LC-c serine mutant (2kDa, 5kDa, 10kDa and 20kDa) was adsorbed onto a separate ELISA plate and then probed with polyclonal anti-TeNT antibodies pre-incubated with each of four concentrations (10. mu.g/mL, 1. mu.g/mL, 0.1. mu.g/mL and 0.01. mu.g/mL) of TeNT-LC-c serine mutant antigen.

FIG. 30 includes four photographs showing local limb twitching in the hindlimb of female C57BL/6 mice receiving PEG-TeNT-LC-HC injection. A and B show local limb twitches in naive mice. C shows no limb spasm after injection of 100 units of TeNT in tetanus toxoid vaccinated mice, while D shows persistent limb spasm after injection of 80 units of PEG-TeNT LC-HC 20kDa in tetanus toxoid vaccinated mice.

FIG. 31 includes two dot plots showing the level of clinical tetanus exhibited in the hindlimb of female C57BL/6 mice after injection of defined number of units of TeNT, PEG-TeNT-LC-HC 20kDa, TeNT-LC-C serine mutant, PEG-TeNT-LC-C serine mutant 2kDa, or PEG-TeNT-LC-C serine mutant 20 kDa. A) Mice were injected with TeNT or PEG-TeNT-LC-HC 20 kDa. B) Mice were injected with TeNT, a TeNT-LC-c serine mutant, a PEG-TeNT-LC-c serine mutant of 2kDa or a PEG-TeNT-LC-c serine mutant of 20 kDa. 1 unit is the minimum amount of toxin required to produce a fourth phase tic within 24 hours in naive mice.

Figure 32 includes two line graphs showing the progression of clinical tics in mice vaccinated with tetanus toxoid vaccine. A shows the progression of twitches after injection of 4000 units of TeNT bait in the presence or absence of inactivated TeNT bait. B shows the progression of tics after injection of 40 units of TeNT-PEG 20kDa in the presence or absence of inactivated TeNT decoy. In both cases, the presence of the bait significantly facilitated progress.

Figure 33 is a boxplot showing the effect of increasing the dose of tetanus toxin on the Respiratory Disturbance Index (RDI) of uk bovine dogs treated according to example 23. The black bars represent the median RDI of six studies performed at each tetanus toxin dose. The shaded boxes represent the interquartile range and the brackets represent the minimum and maximum RDI samples at each dose. Compared to RDI after placebo administration, an increase in tetanus toxin after 10IU/kg administration was associated with a decrease in RDI (P ═ 0.043; Wilcoxon Signed Ranks test). The median values for placebo and 10IU/kg are additionally indicated by o and x, respectively.

Detailed Description

The present invention relates to TeNT and PEG-TeNT molecules that evade immunity (FIG. 1), compositions thereof, and their therapeutic and cosmetic uses. In one embodiment, the invention relates to the treatment of hypotonia, optionally obstructive sleep apnea.

Described herein are the use of certain modified TeNT and a combination of TeNT and bait (inactive TeNT) to treat hypotonia in a subject having a protective immune response against tetanus toxoid. To achieve this, active TeNT was modified by adding PEG, introducing specific mutations, or a combination thereof for delivering bioactive compounds capable of increasing muscle tone in tetanus immunized patients.

The activity can be demonstrated by administering a unit-determined dose of the modified toxin or formulation, wherein at the same unit dose, TeNT will not exhibit activity in the vaccinated subject. Based on the analysis of the three-dimensional structure of the TeNT, the introduction of specific surface mutations to target the attached PEG molecules allows masking of specific TeNT epitopes known to be targeted by protective antibody responses in vaccinated subjects. The combination of pegylation, site-directed mutagenesis, and the bait molecule preparation greatly enhances the effect of the molecule in increasing muscle tension in vaccinated mammalian models relative to the equivalent unit of TeNT administered.

US 2002/0197278 discloses a series of pegylated botulinum toxins for the treatment of inappropriate muscle contraction disorders and indicates that TeNT can be used as a replacement for botulinum toxins. However, TeNT cannot be used to treat muscle contraction. Furthermore, since the disclosed method does not comprise site-directed masking of epitopes, the so-called invention of US 2002/0197278 seems to be unrealized and, to the knowledge of the inventors of the present application, the three-dimensional structure and epitope identification required for the deliberate masking of TeNT epitopes is unknown at the priority date of US 2002/0197278.

Wan et al are concerned with the effect of pegylation on the anti-PEG immune response resulting from administration of pegylated proteins, but have not been exploited for any treatment. Although Wan et al disclose that pegylated tetanus toxoid exhibits reduced immunogenicity relative to non-pegylated tetanus toxoid, Wan et al do not suggest therapeutically relevant molecules or formulations. Furthermore, pegylation of tetanus toxoid is not associated with modification of active TeNT, since tetanus toxoid is a biologically inactive TeNT used for vaccination, which may be produced by formaldehyde cross-linking of TeNT. That is, the pegylated or unpegylated tetanus toxoid has no combination of enzymatic, binding, and translocation activities of active TeNT.

WO 2016/001762 a1 relates only to the TeNT c fragment, which is a molecule with no specific activity other than binding neurotransmitters and entering neurons.

Thus, there is a need for a TeNT-based treatment that avoids pre-existing immunity against TeNT in subjects vaccinated with tetanus toxoid. Disclosed herein are solutions to this problem, provided in part by masked active and therapeutically relevant pegylated TeNT.

Tetanus neurotoxin (TeNT)

TeNT was about 150kDa and was expressed by the tetX gene. A codon optimized nucleic acid sequence corresponding to the coding region for tetX, but lacking the initiating methionine codon, is provided in the vector sequence of FIG. 3(SEQ ID NO: 2). TeNT is expressed as a protein that is post-translationally cleaved, i.e., the initiating methionine is first removed and then cleaved into two parts: a 50kDa light chain (LC or A chain) derived from the N-terminus of the uncleaved protein and a 100kDa heavy chain (HC or B chain) derived from the C-terminus of the uncleaved protein. The two chains are linked by interchain disulfide bonds, which is critical for neurotoxicity. The 1314 amino acid sequence of mature TeNT is provided in FIG. 2 (SEQ ID NO: 1).

LC has zinc endopeptidase activity and attacks the Vesicle Associated Membrane Protein (VAMP) necessary for vesicle fusion with the membrane, thereby preventing neurotransmitter release.

Upon digestion with papain, HC can be cleaved into two domains of 50kDa each: an N-terminal translocation domain designated HN; and a C-terminal ganglioside (membrane) binding domain designated as fragment C (C). TeNT lacking c is referred to herein as LC-HN.

c carry two polysialic ganglioside binding sites and bind to polysialic gangliosides on neuronal membranes (GD2 GD1b and GT1 b). Thus, c mediates binding of the TeNT to the presynaptic membrane of the surrounding motor axons and helps the TeNT to move across this membrane into the neuron.

Amino acid sequence:

the amino acid sequence of TeNT lacking the initiating methionine is provided in FIG. 2 (SEQ ID NO: 1);

the amino acid sequence of HC is provided in FIG. 5(SEQ ID NO: 3);

the amino acid sequence of c is provided in FIG. 6(SEQ ID NO: 4); and is

The amino acid sequence of LC-HN is provided in FIG. 9 (SEQ ID NO: 6).

Codon-optimized nucleic acid sequences and vector maps of vectors encoding:

c is provided in FIG. 7(SEQ ID NO:5) and FIG. 8; and is

LC-HN are provided in FIG. 10(SEQ ID NO:7) and FIG. 11.

As used herein, "TeNT" is used to refer to the entire TeNT molecule consisting of a heavy chain and a light chain. Subdomains and fragments are referred to herein by their abbreviations: light chain "LC"; heavy chain "HC"; heavy chain N-terminal domain "HN"; heavy chain fragment c "c"; the light chain plus heavy chain N-terminal domain "LC-HN" (i.e., TeNT molecule lacking c). In the case where any subdomain or fragment is pegylated, the prefix PEG: PEG-LC; PEG-HC; PEG-HN; PEG-c; PEG-LC-HN. In a complete TeNT molecule comprising a pegylated fragment or subdomain, the prefix PEG is used and indicates the pegylated subdomain or fragment: PEG-TeNT-LC; PEG-TeNT-HC; PEG-TeNT-LC-HC; PEG-TeNT-HN; PEG-TeNT-c; PEG-TeNT-LC-c; PEG-TeNT-LC-HN, and the like.

It will be appreciated that subdomains and fragments are not interchangeable for the complete TeNT, as they have specific functions.

The TeNT disclosed herein may be active or inactive. Active TeNT has the same biological activity as natural TeNT. Inactive TeNT lacks one or more of the activities of native TeNT. In one embodiment, inactive TeNT does not block the release of inhibitory neurotransmitters. Inactive TeNT of the present disclosure may also be referred to as "bait". Inactive TeNT includes tetanus toxoid. In one embodiment, the inactive TeNT is an inactive TeNT as disclosed herein.

In one embodiment, two or more TeNT may be conjugated.

In another embodiment, TeNT is not conjugated. In one embodiment of the compositions, methods, and uses disclosed herein, the first PEG-TeNT is not conjugated to the second TeNT.

The PEG-TeNT that evades immunity disclosed herein and depicted in fig. 1 includes:

TeNT with PEGylated fragment c (PEG-TeNT-c) (FIG. 1A);

TeNT with PEGylated heavy chain (PEG-TeNT-HC) (FIG. 1B);

TeNT (PEG-TeNT-LC-C) with PEGylated light chain and PEGylated fragment C (FIG. 1C); and

fully pegylated TeNT (PEG-TeNT-LC-HC) (FIG. 1D).

PEG-TeNT-c advantageously evades the pre-existing immune response of the adaptive immune system in vaccinated subjects. In one embodiment, PEG-TeNT-c provides a first order treatment to be used until efficacy is reduced.

PEG-TeNT-HC and PEG-TeNT-LC-c advantageously evade pre-existing immune responses of the adaptive immune system in vaccinated subjects and also evade immune responses of the adaptive immune system elicited by repeated exposure to PEG-TeNT-c.

In one embodiment, PEG-TeNT-HC and PEG-TeNT-LC-c together provide a second level of treatment to be used until their efficacy is reduced.

The PEG-TeNT-LC-HC advantageously evades pre-existing immune responses of the adaptive immune system and also evades immune responses of the adaptive immune system elicited by repeated exposure to PEG-TeNT-c and to PEG-TeNT-HC plus PEG-TeNT-LC-c in vaccinated subjects.

In one embodiment, PEG-TeNT-LC-HC provides a third level of treatment to be used until its efficacy is reduced.

Also disclosed are TeNT with a pegylated light chain (PEG-TeNT-LC), TeNT with pegylated LC and HN (PEG-TeNT-LC-HN), and TeNT with pegylated HN (PEG-TeNT-HN).

Those skilled in the art will appreciate that the specific combinations of PEG-TeNT and the order of treatment using those PEG-TeNT combinations may vary.

Polyethylene glycol (PEG)

PEG can be conjugated, for example, to the following amino acid residues in TeNT: lysine (e.g., amino-pegylation), cysteine (e.g., thiol-pegylation, and bridging pegylation), histidine, arginine, aspartic acid, asparagine (e.g., N-glycosyl-pegylation), glutamic acid, glutamine (e.g., transglutaminase-mediated pegylation), serine (e.g., O-glycosyl-pegylation), threonine (e.g., O-glycosyl-pegylation), or tyrosine residue. Examples of pegylation also include N-terminal pegylation and C-terminal pegylation.

Pegylation can be achieved by reacting PEG with functional groups that are hydroxyl reactive, such as anhydrides, acid chlorides, chloroformates, and carbonates. Alternatively, pegylation can be achieved with functional groups such as aldehydes, esters, and amides.

The PEG may be linear or branched.

The PEG may be a modified PEG, such as poly [ oligo (ethylene glycol) methyl ether methacrylate ] (POEGMA).

The pegylation may be site-specific pegylation.

In one embodiment, the surface serine residue of the TeNT or TeNT fragment is mutated to a surface cysteine residue to facilitate targeted PEG conjugation at the immunogenic epitope. In this context, mutation is synonymous with substitution, e.g., serine is substituted with cysteine. Such mutations or substitutions include any combination of one or more of the following: S81C; S120C; S144C; S248C; S335C; S428C; S600C; S963C; S1041C; S1155C; and S1187C.

Functional groups for heterobifunctional PEGs include maleimide, vinyl sulfone, pyridyl disulfide, amines, carboxylic acids, and NHS esters.

In one embodiment, PEG is conjugated to TeNT using carbodiimide-EDC and sulfo-NHS with carboxy-amine cross-linking.

PEG-TeNTs comprising PEG of different molecular weights conjugated to different subdomains or fragments of the TeNT are also contemplated by the invention.

For example, PEG can be conjugated or linked to TeNT of the present disclosure at 4 ℃ to 25 ℃ for 2 hours to 6 hours. In one embodiment, PEG is conjugated to TeNT for 6 hours at room temperature.

Indications of

As used herein, "hypotonia" refers to any condition including involuntary muscle weakness that can be treated by inhibiting inhibitory neurotransmitters, such as GABA or glycine. Thus, "hypotonia" includes a decrease in muscle tone secondary to a decrease in nervous system driving force or other causes and disorders of decreased or insufficient muscle tone, strength, or nervous system driving force. Thus, in one embodiment, the hypotonia may be a neurotonia.

Hypotonic conditions that may be treated with PEG-TeNT, compositions, or methods according to the present disclosure include obstructive sleep apnea, snoring, scoliosis, strabismus due to muscle relaxation, ptosis, Horner's syndrome, muscle atrophy, impaired muscle nerve function, Amyotrophic Lateral Sclerosis (ALS), motor neuron disease, any myopathy, multiple sclerosis, Parkinson's disease, myasthenia gravis, decreased facial muscle tone, optional blepharoptosis, flaccid paralysis or weakness due to any etiology of any skeletal or smooth muscle, respiratory muscle weakness due to any etiology, including post-ventilator weakness; muscle weakness caused by trauma or postural problems caused by muscle relaxation, muscle relaxation or weakness in the pelvic floor or relaxation of the nasal or upper respiratory tract.

Other conditions that may be treated with PEG-TeNT, compositions, or methods according to the present disclosure include muscle atrophy, muscular dystrophy, decreased muscle mass, nasal congestion, impotence, hair loss, hypotension, temporomandibular joint syndrome, torticollis, neck pain, intramuscular nerve regeneration, migraine, headache, achalasia, obesity, colonic spasm, anal fissure, tissue or organ affected by gastric acid, prostatic hypertrophy, rhinorrhea, salivation, irritation of the pulmonary mucosa, psoriasis, immune tolerance, immune response.

Where the disorder to be treated according to the present disclosure is not a hypotonic disorder per se, the symptoms of the disorder may be alleviated by increasing muscle tone by treatment with TeNT of the present disclosure.

Cosmetic applications of PEG-TeNT may include tightening of abdominal muscles, tightening of pectoral muscles, tightening of gluteus maximus, tightening of skeletal muscles, or treatment of facial ptosis caused by muscle relaxation.

Smooth muscle, skeletal muscle, tissue or organ that may be treated with PEG-TeNT, compositions or methods according to the present disclosure include upper esophagus, esophageal wall, esophageal sphincter, lower esophageal sphincter, anal sphincter, bladder sphincter, vaginal sphincter, pyloric sphincter, audi sphincter, ileocecal sphincter, pelvic floor muscle, vaginal wall muscle, prostate, submandibular gland, parotid gland, sublingual gland, the small salivary gland of the oral mucosa, vocal cords, facial muscle, masticatory muscle, scalp muscle, pectoral muscle, dorsal muscle, upper limb muscle, forearm muscle, lower limb muscle, hand muscle, foot muscle, stomach wall muscle, colon wall muscle, neck muscle, throat dilator muscle, masseter, intra-pterygeus muscle, pterygopal muscle, geniohyoid muscle, palatoglossus muscle, levator muscle, stylopharyngeal muscle, styloglossus muscle, mylohyoid muscle, styloglossus, glossoglossus, hyoid muscle, glossoglossus muscle, and the like, The digastric muscle, sternocleidomastoid muscle, trapezius muscle, temporal muscle, cricopharyngeal muscle, uterine muscle and cervix, gastric innervation, intranasal mucosa, pulmonary mucosa, skin, thymus, bone, coronary arteries, pulmonary smooth muscle, and cardiac muscle.

Compositions and applications

The composition of the present disclosure may be a therapeutic composition or a cosmetic composition. That is, the composition may be used for therapeutic or cosmetic purposes.

The term "therapeutic composition" or "cosmetic composition" as used herein refers to a composition comprising TeNT that inhibits or treats hypotonia in a subject as described herein. The composition has been formulated for administration to a subject. In one embodiment, the composition is sterile. In one embodiment, the composition is pyrogen-free. The composition may comprise a pharmaceutically acceptable carrier. Preferably, the composition is manufactured according to Good Laboratory Practice (GLP) or Good Manufacturing Practice (GMP).

The TeNT of the present disclosure can be administered at doses up to 10mg/kg or higher. The TeNT of the present disclosure may be administered at the following doses: about 1fg/kg, about 5fg/kg, about 10fg/kg, about 50fg/kg, about 100fg/kg, about 500fg/kg, about 1pg/kg, about 5pg/kg, about 10pg/kg, about 50pg/kg, about 100pg/kg, about 500pg/kg, about 1ng/kg, about 2ng/kg, about 3ng/kg, about 4ng/kg, about 5ng/kg, about 6ng/kg, about 7ng/kg, about 8ng/kg, about 9ng/kg, about 10ng/kg, about 11ng/kg, about 12ng/kg, about 13ng/kg, about 14ng/kg, about 15ng/kg, about 16ng/kg, about 17ng/kg, about 18ng/kg, about 19ng/kg, about 20ng/kg, about 30ng/kg, About 40ng/kg, about 50ng/kg, about 60ng/kg, about 70ng/kg, about 80ng/kg, about 90ng/kg, about 100ng/kg, about 200ng/kg, about 300ng/kg, about 400ng/kg, about 500ng/kg, about 600ng/kg, about 700ng/kg, about 800ng/kg, about 900ng/kg, about 1 μ g/kg, about 5 μ g/kg, about 10 μ g/kg, about 50 μ g/kg, about 100 μ g/kg, about 500 μ g/kg, about 1mg/kg, or about 10 mg/kg. The TeNT of the present disclosure may be administered within any range of any of the dosages listed above.

The TeNT of the present disclosure can be administered at doses up to 1000IU/kg or higher. The TeNT of the present disclosure may be administered at the following doses: about 0.1IU/kg, about 0.2IU/kg, about 0.3IU/kg, about 0.4IU/kg, about 0.5IU/kg, about 0.6IU/kg, about 0.7IU/kg, about 0.8IU/kg, about 0.9IU/kg, about 1IU/kg, about 2IU/kg, about 3IU/kg, about 4IU/kg, about 5IU/kg, about 6IU/kg, about 7IU/kg, about 8IU/kg, about 9IU/kg, about 10IU/kg, about 11IU/kg, about 12IU/kg, about 13IU/kg, about 14IU/kg, about 15IU/kg, about 16IU/kg, about 17IU/kg, about 18IU/kg, about 19IU/kg, about 20IU/kg, about 30IU/kg, about 40IU/kg, about 50IU/kg, about 60IU/kg, About 70IU/kg, about 80IU/kg, about 90IU/kg, about 100IU/kg, about 200IU/kg, about 300IU/kg, about 400IU/kg, about 500IU/kg, about 600IU/kg, about 700IU/kg, about 800IU/kg, about 900IU/kg, about 1000 IU/kg. The TeNT of the present disclosure may be administered within any range of any of the dosages listed above.

In a composition comprising two PEG-TeNTs, for example, a composition comprising a first PEG-TeNT (PEG-TeNT-HC) in which TeNT-HC is pegylated and a second PEG-TeNT (PEG-TeNT-LC-c) in which TeNT-LC is pegylated, the ratio of the first PEG-TeNT to the second PEG-TeNT can vary. For example, the ratio of the first PEG-TeNT to the second PEG-TeNT may be about 1000:1, about 500:1, about 100:1, about 50:1, about 10:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:50, about 1:100, about 1:500, or about 1: 1000.

The composition may comprise any combination of TeNT and is not limited to the combination of PEG-TeNT-HC and PEG-TeNT-LC-c. The composition may comprise: PEG-TeNT-c and PEG-TeNT-HC; PEG-TeNT-c and PEG-TeNT-LC-c; PEG-TeNT-c and PEG-TeNT-LC-HC; PEG-TeNT-HC and PEG-TeNT-LC-HC; and PEG-TeNT-LC-c and PEG-TeNT-LC-HC. Also disclosed is a composition comprising: PEG-TeNT-c, PEG-TeNT-HC and PEG-TeNT-LC-c; PEG-TeNT-c, PEG-TeNT-HC and PEG-TeNT-LC-HC; PEG-TeNT-c, PEG-TeNT-LC-c and PEG-TeNT-LC-HC; PEG-TeNT-HC, PEG-TeNT-LC-c and PEG-TeNT-LC-HC; and PEG-TeNT-c, PEG-TeNT-HC, PEG-TeNT-LC-c and PEG-TeNT-LC-HC. Any TeNT may be substituted in the composition, and any composition may further comprise PEG-TeNT-LC, PEG-TeNT-LC-HN, and/or PEG-TeNT-HN.

In one embodiment, the composition further comprises inactivated TeNT that acts as a bait for anti-TeNT antibodies generated by prior exposure to TeNT, e.g., by vaccination. Relative to SEQ ID NO:1, the inactivated TeNT may comprise: R1225K; R1225E; W1228A; W1288Y; W1288F; W1288L; R1225K and W1288A; R1225K and W1288Y; R1225E and W1288Y; R1225K and W1288F; R1225E and W1288F; R1225K and W1288L; R1225E and W1288L; r1225 del; w1288 del; or R1225del and W1288 del; E270A; Y374A; E270A and Y374A; e270 del; y374del or combinations thereof. In one embodiment, the second TeNT comprises an inactivated TeNT comprising R1225E, W1288A, E270A, and Y374A.

In a composition comprising at least one PEG-TeNT and a bait TeNT, the bait TeNT will be in molar excess relative to the PEG-TeNT. For example, the ratio of bait TeNT to PEG-TeNT can be about 10⁶:1；10⁵:1、10⁴1, 1000:1, about 500:1, about 400:1, about 300:1, about 200:1, about 100:1, about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1: 1.

The PEG-TeNT of the present disclosure may be administered once, twice, or three times per week; once, twice or three times monthly; once, twice or three times a quarter; once, twice or three times every 6 months; or once, twice or three times a year.

The PEG-TeNT may be administered in a single dose, divided doses, or multiple doses. In the case of a paired muscle, PEG-TeNT may be administered to one muscle of the pair on a single side or to both muscles of the pair on both sides.

As an alternative to a combination comprising two or more PEG-TeNT of the present disclosure, the two or more PEG-TeNT may be administered in combination, either sequentially or simultaneously.

The PEG-TeNT can be administered topically to the subject by any suitable method, for example, by injection, surgical implantation, topical administration, or intranasal administration. In one embodiment, TeNT is administered intramuscularly by injection into the affected muscle.

The PEG-TeNT will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this context include the particular type of hypotonia being treated, the particular subject being treated, the clinical condition of the subject, the site of administration, the method of administration, the timing of administration, and other factors known to physicians, including dentists. A therapeutically effective amount of PEG-TeNT to be administered will depend on such considerations.

Pharmaceutically acceptable carriers include water, buffered water, saline solutions, e.g., physiological saline or balanced saline solutions, such as Hank's balanced solution or early's balanced solution, glycine and hyaluronic acid.

The composition may be formulated for intramuscular administration. Compositions for intramuscular administration may comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, solvents, diluents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol), carboxymethylcellulose and mixtures thereof, vegetable oils (such as olive oil), injectable organic esters (such as ethyl oleate).

The compositions may include a penetration enhancer to facilitate their TeNT delivery. The penetration enhancer may include fatty acids such as oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, reciline, glycerol monooleate, glycerol dilaurate, caprylic acid, arachidonic acid, glycerol-1-monodecanoate, monoglycerides and diglycerides, and physiologically acceptable salts thereof.

The composition may also include chelating agents such as ethylenediaminetetraacetic acid (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate, homovanillic acid salts).

Also provided is an article of manufacture and/or a kit comprising a container comprising a PEG-TeNT or a composition comprising a PEG-TeNT. The container can be a bottle, vial, or syringe containing PEG-TeNT or the composition (optionally in unit dosage form). For example, the PEG-TeNT or composition may be in the form of an injectable solution in a disposable container, optionally a syringe. The article of manufacture and/or kit may further comprise printed instructions and/or labeling, etc., instructing the subject to be treated according to the methods disclosed herein.

The term "therapeutically effective amount" refers to an amount of PEG-TeNT effective to treat hypotonia in a subject.

The terms "treatment", "treating" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, reduce or ameliorate hypotonia or to slow down (lessen) the progression of hypotonia in a subject. Subjects in need of treatment include subjects already suffering from hypotonia as well as subjects in whom hypotonia is to be prevented or ameliorated.

The terms "preventing", "prevention", "preventive" or "preventative" refer to avoiding, hindering, defending or preventing the occurrence of hypotonia. A subject in need of prophylaxis may be susceptible to hypotonia.

The term "improve" or "amelioration" refers to the reduction, or elimination of hypotonia.

Hypotonia can be quantified. Hypotonia can be quantified on a semi-quantitative scale, such as 0 to 5, where 0 represents absence, 1 to 4 represents a discernible increase in severity, and 5 represents maximum severity. Alternatively, hypotonia can be quantified as a binary event, i.e., the presence or absence (0 or 1). Other semi-quantitative scales will be apparent to those skilled in the art. In another embodiment, the hypotonia may be quantified on a quantitative scale, for example using a dynamometer.

Any quantification of hypotonia can be compared to a control, e.g., a healthy control subject that does not receive PEG-TeNT, a diseased control subject that receives treatment for hypotonia, but is not treated with PEG-TeNT, or a population of humans.

Treatment of hypotonia by administration of PEG-TeNT may be a reduction in hypotonia of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100%.

The term "subject" as used herein may refer to a mammal. The mammal may be a primate, particularly a human, or may be a domestic, zoo or companion animal. While it is specifically contemplated that the PEG-TeNT, compositions and methods disclosed herein are suitable for use in medical treatment of humans, it is also suitable for use in veterinary treatment, including the treatment of livestock, such as horses, cattle and sheep; companion animals such as dogs and cats; or zoo animals such as felines, canines, bovines, and ungulates.

Unless defined otherwise in the present specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs by reference to the disclosure.

It is noted that the term "a/an" refers to one or more, e.g., "a TeNT" should be understood to mean one or more TeNT. Thus, the terms "a/an", "one or more" and "at least one" are used interchangeably herein.

In the appended claims and this specification of the invention, unless the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

The term "about" as used herein encompasses a range of values for a given quantity that is ± 25% different in magnitude from the quantity. In other embodiments, the term "about" encompasses a range of values for a given quantity that varies from the magnitude of the quantity by ± 20%, ± 15%, ± 10%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1%. For example, in one embodiment, "about 3 grams" means a value of 2.7 grams to 3.3 grams (i.e., 3 grams ± 10%), and the like.

Similarly, the time or duration of the event may vary by at least 25%. For example, while a particular event may be disclosed as lasting for one day in one embodiment, the event may last for more than one day or less than one day. For example, "a day" may include a period of time from about 18 hours to about 30 hours. In other embodiments, the time period may vary by 20%, ± 15%, ± 10%, ± 5%, ± 4%, ± 3%, ± 2% or ± 1% of the time period.

Examples

Example 1: preparation of PEG-TeNT-c (FIG. 1A)

In this example, the surface serine residue of TeNT-c was mutated to a surface cysteine residue to facilitate targeted PEG conjugation at an immunogenic epitope to produce the molecule of fig. 1A. The mutation will be: S963C, S1041C, S1155C and S1187C.

The gene for TeNT having the surface serine → cysteine substitutions S963C, S1041C, S1155C and S1187C will be synthesized by commercial suppliers (e.g., Integrated DNA Technologies). The gene was subcloned into the pRSET-A mutexpression vector by restriction digestion to add a 6 × histidine tag from the vector to the N-terminus of the mutein.

TeNT comprising S963C, S1041C, S1155C and S1187C will be expressed, pegylated, and purified according to example 5 to produce PEG-TeNT-c.

Example 2: preparation of PEG-TeNT-HC (FIG. 1B)

In this example, the surface serine residue of TeNT-HC was mutated to a surface cysteine residue to facilitate targeted PEG conjugation at the immunogenic epitope to produce the molecule of fig. 1B. The mutations will be: S600C, S963C, S1041C, S1155C and S1187C (FIG. 24, SEQ ID NO: 16).

Genes for TeNT having surface serine → cysteine substitutions S600C, S963C, S1041C, S1155C and S1187C will be synthesized by commercial suppliers (e.g., Integrated DNA Technologies, inc.). The gene was subcloned into the pRSET-A mutexpression vector (FIG. 25, SEQ ID NO:17, FIG. 26) by restriction digestion to add a 6 × histidine tag from the vector to the N-terminus of the mutein.

TeNT comprising S600C, S963C, S1041C, S1155C and S1187C will be expressed, pegylated, and purified according to example 5 to produce PEG-TeNT-HC.

Example 3: preparation of PEG-TeNT-LC-C (FIG. 1C)

In this example, surface serine residues of LC and C were mutated to surface cysteine residues to facilitate targeted PEG conjugation at immunogenic epitopes to produce the molecule of fig. 1C. The mutations are: S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C and S1187C (FIG. 21, SEQ ID NO: 14).

Genes for TeNT having surface serine → cysteine substitutions S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C and S1187C in LC and c were synthesized by commercial suppliers (Integrated DNA Technologies). The gene was subcloned into the pRSET-A mutexpression vector (FIG. 22, SEQ ID NO:15, FIG. 23) by restriction digestion to add a 6 × histidine tag from the vector to the N-terminus of the mutein.

TeNT expression comprising S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C and S1187C was pegylated and purified according to example 5 to produce PEG-TeNT-LC-c.

Endotoxin removal

Endotoxin was removed from the TeNT-LC-c serine mutant according to example 4.

Attachment of PEG to cysteine residues.

1. A molar excess of PEG-maleimide is combined with the serine mutant TeNT-LC-c (0.5mg/mL-2mg/mL) in PBS at pH 6.5-7.5, wherein the PEG has about 2kDa, about 5kDa, about 10kDa, or about 20 kDa.

2. The ligation was performed at room temperature for 6 hours.

3. Excess PEG was removed by size exclusion chromatography.

Trypsin digestion activation of proteins

Trypsin digestion was performed according to example 4 to activate the TeNT-LC-c serine mutant.

Example 4: preparation of PEG-TeNT-LC-HC (FIG. 1D) (method 1)

Preparation of TeNT

1. Coli BL21 DE3 pLysS strain was electro-transformed with pRSET-TeNT vector (FIGS. 3 and 4) and grown overnight at 37 ℃ on LB agar with selection antibiotics (ampicillin and chloramphenicol).

2. 200mL of pre-induction broth was inoculated with 1 colony from the selection plate. pre-induction broth pH 7.2-7.4 contains: 1.2% tryptone; 2.4% yeast extract; 2% glucose; 0.4% of glycerol; 17mM KH₂PO₄；72mM K₂HPO₄(ii) a And selection antibiotics (ampicillin and chloramphenicol).

3. The cultures were incubated overnight at 30 ℃ with rapid shaking.

4. The overnight culture was harvested by centrifugation at 4000g for 10 minutes.

5. The pellet was resuspended in 200mL of expression broth (pH 7.2-7.4) containing: 1.2% tryptone; 2.4% yeast extract; 0.4% of glycerol; 1mM IPTG; 17mM KH₂PO₄；72mM K₂HPO₄(ii) a 100 mu g/mL ampicillin; and 10. mu.M ZnCl₂。

6. The protein was expressed at 30 ℃ for 6 hours with rapid shaking.

7. Cells were harvested by centrifugation at 4500g for 15 min and the pellet resuspended in 30mL TBS (pH 8) containing 20mM imidazole.

8. Cells were lysed by sonication.

9. The cell lysate was clarified by centrifugation at 4500g for 20 min and filtered through a 0.45 μm filter.

10. The protein was purified by His-tag affinity chromatography using the AKTA pure 25FPLC system (general electric company (GE)).

11. The purified protein was buffer exchanged with PBS using size exclusion chromatography followed by a second stage purification by gel filtration using AKTA pure 25FPLC and Superdex 200 plus 10/300GL column.

Endotoxin removal

1. 0.5mL of endotoxin removal spin column was equilibrated to room temperature.

2. The bottom plug was removed, the cap was loosened, the column was placed in a 15mL tube, and then centrifuged at 500g for 1 minute to remove the solution from the column. The solution was discarded.

3. The bottom plug was replaced, the cap removed, 0.2N NaOH in 95% ethanol was added to the resin, the cap replaced, the column was inverted several times to resuspend the resin, and then incubated at room temperature for 1-2 hours.

4. The bottom plug was removed, the cap was loosened, the column was placed in a 15mL tube, and then centrifuged at 500g for 1 minute to remove the solution from the column. The solution was discarded.

5. The column bottom plug was replaced, the column cap was removed, endotoxin-free 2M NaCl was added to the resin, the column cap was replaced, and the column was inverted several times to resuspend the resin.

6. The bottom plug was removed, the cap was loosened, the column was placed in a 15mL tube, and then centrifuged at 500g for 1 minute to remove the solution from the column. The solution was discarded.

7. The bottom plug was replaced, the cap was removed, endotoxin-free ultrapure water was added to the resin, the cap was replaced, and the column was inverted several times to resuspend the resin.

8. The bottom plug was removed, the cap was loosened, the column was placed in a 15mL tube, and centrifuged at 500g for 1 minute to remove the solution from the column. The solution was discarded.

9. The bottom plug was replaced, the cap was removed, endotoxin-free phosphate buffer was added to the resin, the cap was replaced, and the column was inverted several times to resuspend the resin.

10. The bottom plug was removed, the cap was loosened, the column was placed in a 15mL tube, and centrifuged at 500g for 1 minute to remove the solution from the column. The solution was discarded.

11. The column was washed twice more with phosphate buffer and the eluate was discarded.

12. The bottom plug was replaced, the cap removed, the sample applied to the resin, the cap replaced, and the column inverted several times to resuspend the resin.

13. The column was incubated at 4 ℃ for at least 1 hour with inverted mixing.

14. The bottom plug was removed, the cap was loosened, the column was placed in a 15mL tube without endotoxin, and centrifuged at 500g for 1 minute to remove the solution from the column. The sample was retained.

15. The endotoxin removal procedure was repeated using a regenerated spin column until the endotoxin level in the sample was at an equivalent or lower level so that all doses would contain less than 5EU units of endotoxin per kg of subject.

Preparation of PEG-TeNT-LC-HC

1. Mu. mol of purified TeNT and 0.5mmol of mpeg-NHS (SC) (Nanocs Co.) were combined with one of 2kDa, 5kDa, 10kDa, 20kDa or 30kDa PEG in a total volume of 500. mu.L of PBS (pH 7.4).

2. The sample was mixed at room temperature for 3 hours.

3. Excess PEG was removed by size exclusion chromatography.

Trypsin digests proteins into active form

1. 1mg of protein was dissolved in 0.5mL of digestion buffer containing 0.1M NH₄HCO₃Buffer (pH 8.0) or 0.1M Tris buffer (pH 8.5).

2. 0.10mL to 0.25mL of immobilized TPCK trypsin was washed with 3X 500. mu.L of digestion buffer. The gel was separated from the buffer by centrifugation after each wash.

3. The gel was resuspended in about 0.2mL of digestion buffer.

4. Immobilized TPCK trypsin was added to the protein sample.

5. The reaction mixture was incubated in a rapid shaking incubator at 37 ℃ for 2 to 18 hours.

6. The immobilized TPCK trypsin was isolated by centrifugation.

Example 5: preparation of PEG-TeNT-LC-HC (FIG. 1D) (method 2)

In this example, the surface serine residue of TeNT-LC-HC was mutated to a surface cysteine residue (S → C mutant) to facilitate targeted PEG conjugation at the immunogenic epitope. The TeNT mutation relative to SEQ ID NO 1 is: S81C; S120C; S144C; S248C; S335C; S428C; S600C; S963C; S1041C; S1155C; and S1187C.

Preparation of serine mutant TeNT-LC-HC

Coli BL21(DE3) pLysS strain was electrotransformed with the vector pRSET-TeNT (FIGS. 19 and 20) encoding the amino acid sequence of FIG. 18 (SEQ ID NO:12) comprising the S → C mutation. TenT-LC-HC containing the S → C mutation was expressed and purified according to example 4, with an increase of 15 minutes of treatment with 0.5mM DTT between step 10 and step 11.

Endotoxin removal

Endotoxin was removed from the TeNT-LC-HC serine mutant according to example 4. Attachment of PEG to cysteine residues.

1. Combining a molar excess of PEG-maleimide with a serine mutant TeNT-LC-HC (0.5mg/mL-2mg/mL) in PBS at pH 6.5-7.5, wherein the PEG has about 2kDa, about 5kDa, about 10kDa, or about 20 kDa.

2. The ligation was performed at room temperature for 6 hours.

3. Excess PEG was removed by size exclusion chromatography.

Trypsin digestion activation of proteins

Trypsin digestion was performed according to example 4 to activate the TeNT-LC-HC serine mutants.

Example 6: inactive bait TeNT

In this example, non-pegylated recombinant c with inactive R1225E and W1288A amino acid substitutions relative to SEQ ID No. 1 in the ganglioside binding region was generated. Inactivated c was combined with equimolar amounts of non-pegylated LC-HN to produce inactive decoy TeNT. The resulting inactive TeNT will be used as a decoy for antibody-based neutralization responses in subjects vaccinated with tetanus toxoid vaccine.

Preparation of c comprising R1225E, W1288A

The genes having c of R1225E and W1288A were synthesized by commercial suppliers (Integrated DNA Technologies Co.). The gene was subcloned into the pRSET-A mutexpression vector by restriction digestion to add a 6 × histidine tag from the vector to the N-terminus of the mutein.

Coli BL21(DE3) pLysS strain was electrotransformed with pRSET-TeNT-c vector (FIG. 13(SEQ ID NO:9) and FIG. 14) encoding a c comprising R1225E, W1288A (FIG. 12, SEQ ID NO:8) and allowed to grow overnight at 37 ℃ on LB agar containing selection antibiotics (ampicillin, chloramphenicol). C comprising R1225E, W1288A was expressed and purified according to example 4.

Endotoxin removal

Endotoxin was removed from the purified protein according to example 4.

Preparation of LC-HN

Coli BL21(DE3) pLysS strain was electrotransformed with pRSET-TeNT-LC-HN vector (FIGS. 10 and 11) and grown overnight at 37 ℃ on LB agar with selection antibiotics (ampicillin, chloramphenicol). LC-HN was expressed and purified according to example 4.

Endotoxin removal

Endotoxin was removed from the purified protein according to example 4.

Preparation of LC-HN comprising E270A, Y374A

Coli BL21(DE3) pLysS strain was electrotransformed with a pRSET-TeNT-LC-HN vector encoding the LC-HN comprising E270A, Y374A and allowed to grow overnight at 37 ℃ on LB agar containing the selection antibiotics (ampicillin, chloramphenicol). LC-HN comprising E270A, Y374A was expressed and purified according to example 4.

Endotoxin removal

Endotoxin was removed from the purified protein according to example 4.

Example 7: inactive bait TeNT

The gene of TeNT comprising R1225E and W1288A was synthesized by a commercial supplier (Integrated DNA Technologies). The gene was subcloned into the pRSET-A mutexpression vector by restriction digestion to add a 6 × histidine tag from the vector to the N-terminus of the mutein (FIG. 15(SEQ ID NO:10), FIG. 16(SEQ ID NO:11), and FIG. 17).

Inactivated TeNT comprising R1225E and W1288A was expressed and purified according to example 4.

Endotoxin removal

Endotoxin was removed from the purified protein according to example 4.

Example 8: PEG-TeNT assay

TeNT was prepared according to example 4 and pegylated, then analyzed by SDS-PAGE (fig. 28) and detected by western blot using (a) coomassie blue and (B) polyclonal anti-TeNT antibody. (B) Immunogenicity is shown to be proportional to PEG molecular weight.

Example 9: reduced immunogenicity of PEG-TeNT compared to TeNT

Four PEG-TeNTs, each comprising different molecular weight PEGs (2kDa, 5kDa, 10kDa and 20kDa), were prepared and pegylated according to example 4. PEG-TeNT was then assayed for TeNT by competitive ELISA.

In the first assay (fig. 29A), TeNT was adsorbed onto ELISA plates. Adsorbed TeNT is then probed with a polyclonal anti-TeNT antibody pre-incubated with each of the four PEG-TeNT antigens (2kDa, 5kDa, 10kDa, and 20kDa) at four concentrations (10. mu.g/mL, 1. mu.g/mL, 0.1. mu.g/mL, and 0.01. mu.g/mL). In this assay, a higher response (OD 450nm) indicates a greater affinity for TeNT and, therefore, reduced immunogenicity of PEG-TeNT.

In a second assay (fig. 29B), each PEG-TeNT was adsorbed onto a separate ELISA plate. Each adsorbed PEG-TeNT (2kDa, 5kDa, 10kDa, and 20kDa) was then probed with polyclonal anti-TeNT antibodies pre-incubated with each of the four concentrations (10. mu.g/mL, 1. mu.g/mL, 0.1. mu.g/mL, and 0.01. mu.g/mL) of TeNT antigen. In this assay, a lower response (OD 450nm) indicates greater affinity for TeNT and, therefore, reduced immunogenicity of PEG-TeNT.

This example demonstrates that anti-TeNT antibodies preferentially bind TeNT and that pegylated TeNT has reduced immunogenicity relative to TeNT (i.e., non-pegylated).

Example 10: reduced immunogenicity of PEG-TeNT-LC-c serine mutants compared to TeNT-LC-c serine mutants

TenT-LC-c serine mutants were prepared according to example 5. Four samples of the TeNT-LC-c serine mutant, each containing a different molecular weight of PEG (2kDa, 5kDa, 10kDa and 20kDa), were pegylated according to example 5. The PEG-TeNT-LC-c serine mutant was then assayed for the TeNT-LC-c serine mutant by competitive ELISA.

In the first assay (FIG. 29C), the TeNT-LC-C serine mutant was adsorbed onto an ELISA plate. The adsorbed TeNT serine mutants were then probed with polyclonal anti-TeNT antibodies pre-incubated with each of the four PEG-TeNT serine mutant antigens (2kDa, 5kDa, 10kDa and 20kDa) at four concentrations (10. mu.g/mL, 1. mu.g/mL, 0.1. mu.g/mL and 0.01. mu.g/mL). In this assay, a higher response (OD 450nm) indicates a greater affinity for the TeNT serine mutant and, therefore, reduced immunogenicity of the PEG-TeNT serine mutant.

In a second assay (FIG. 29D), each PEG-TeNT-LC-c serine mutant was adsorbed onto a separate ELISA plate. Each adsorbed PEG-TeNT serine mutant (2kDa, 5kDa, 10kDa, and 20kDa) was then probed with polyclonal anti-TeNT antibodies pre-incubated with each of the four concentrations (10. mu.g/mL, 1. mu.g/mL, 0.1. mu.g/mL, and 0.01. mu.g/mL) of TeNT serine mutant antigen. In this assay, a lower response (OD 450nm) indicates greater affinity for the TeNT serine mutant and, therefore, reduced immunogenicity of the PEG-TeNT serine mutant.

This example demonstrates that anti-TeNT antibodies preferentially bind to TeNT-LC-c serine mutants and that pegylated TeNT-LC-c serine mutants have reduced immunogenicity relative to TeNT-LC-c serine mutants (i.e., non-pegylated).

Example 11: reduced immunogenicity of PEG-TeNT compared to TeNT

A competitive ELISA assay will be performed according to example 9 except that the polyclonal antibodies will be replaced with human serum collected from one or more subjects who have received a booster tetanus toxoid vaccine within the previous 12 months. Antibodies in serum will show greater affinity for TeNT (i.e., non-pegylated TeNT) than for PEG-TeNT.

Example 12: in vivo model

PEG-TeNT-LC-HC was prepared according to example 4 by linking PEG (about 5kDa, about 10kDa, or about 20kDa) to surface-exposed lysine residues of recombinant TeNT.

One or more units of PEG-TeNT-LC-HC in 15. mu.L PBS was injected into the hind limb of female C57BL/6 mice. Each animal showed local limb convulsions within 48 hours of injection (fig. 30A and 30B).

Example 13: in vivo model

PEG-TeNT-LC-HC according to example 4 with 5kDa PEG attached to surface lysine residues of TeNT was combined with c bait (i.e. c inactivated by R1225E and W1288A) in a molar ratio of 1: 10. A15 microliter composition containing 5ng of PEG-TeNT in PBS was injected into the hind limb muscle of female C57BL/6 mice. Each animal showed local limb twitching within 48 hours, and symptom development was indistinguishable from animals treated with PEG-TeNT-LC-HC in the absence of bait.

Example 14

PEG-TeNT-LC-HC containing 5kDa, 10kDa or 20kDa PEG was administered intramuscularly to the hind leg muscle of mice previously vaccinated with tetanus toxoid at 50ng/kg to 500000 ng/kg. An increase in muscle contraction was observed in the injected muscle for up to 3 days, and was greater than the effect observed in mice receiving the same unit of TeNT administration. FIG. 31A reports the results for PEG-TeNT-LC-HC 20 kDa.

Example 15

Compositions comprising TeNT-LC-HC and a 10:1 or 100:1 molar excess of decoy TeNT produced according to example 6 were administered intramuscularly at 50ng/kg to 500000ng/kg to the hind leg muscle of mice previously vaccinated with tetanus toxoid. Increased muscle contraction was observed in the injected muscles for up to 3 days, and was greater than the effect observed in mice receiving TeNT administration (fig. 32A).

Example 16

Compositions containing PEG-TeNT-LC-HC comprising 5kDa, 10kDa or 20kDa PEG and a 10:1 or 100:1 molar excess of decoy TeNT produced according to example 6 were administered intramuscularly at 50ng/kg to 500000ng/kg to the hindleg muscle of mice previously vaccinated with tetanus toxoid. Increased muscle contraction was observed in the injected muscles for up to 3 days, and was greater than the effect observed in mice receiving TeNT administration, and greater than the effect observed in mice receiving PEG-TeNT administration alone (fig. 32B).

Example 17

PEG-TeNT-LC-HC containing 20kDa PEG was administered intramuscularly to the left geniohyoid muscle of a human subject previously vaccinated with tetanus toxoid at 0.01ng/kg to 50000 ng/kg. An increase in muscle contraction will be observed in the injected muscle for up to 2 weeks, and will be greater than the effect observed in the right geniohyoid muscle of the same subject receiving only vehicle administration.

Example 18

A composition containing PEG-TeNT-LC-HC comprising 20kDa PEG and a bait TeNT produced according to example 6 in a 10:1-1000:1 molar excess was administered intramuscularly at 50ng/kg to the left geniohyoid muscle of human subjects previously vaccinated with tetanus toxoid. PEG-TeNT-LC-HC containing 20kDa PEG was administered intramuscularly to the right geniohyoid muscle of the same human subject at 50 ng/kg.

Increased muscle contraction will be observed in both the left and right geniohyoid muscles for up to 2 weeks, but will be greater in the left geniohyoid muscle treated with the composition comprising PEG-TeNT-LC-HC and decoy TeNT compared to PEG-TeNT-LC-HC alone.

Example 19

25ng/kg to 50000ng/kg of PEG-TeNT-c comprising 20kDa PEG will be administered intramuscularly to a bovine cephalic dog of about 30kg, wherein the doses are administered bilaterally separately to the left and right geniohyoid muscles. After administration, animals receiving PEG-TeNT treatment will have reduced Obstructive Sleep Apnea (OSA) compared to animals treated with vehicle alone. Bovine cephalic OSA will be observed once a week and the PEG-TeNT-c dose will be repeated as necessary until efficacy is reduced as determined by recovery of OSA comparable to animals treated with vehicle alone.

Thereafter, 25-50000ng/kg of PEG-TeNT-HC or 25-50000ng/kg of PEG-TeNT-LC-c, each comprising 20kDa PEG, will be administered to the bovine cephalic dog bilaterally separately to the left and right geniohyoid muscles. After administration, OSA will be reduced in animals receiving PEG-TeNT treatment compared to animals treated with vehicle alone. Bovine cephalic OSA will be observed once a week and repeated PEG-TeNT-HC and PEG-TeNT-LC-c doses will be repeated as necessary until efficacy is reduced, as determined by recovery of OSA comparable to animals treated with vehicle alone.

Thereafter, 25-50000ng/kg of PEG-TeNT-LC-HC containing 20kDa PEG will be administered to the bovine cephalic dogs bilaterally separately to the left and right geniohyoid muscles. After administration, OSA will be reduced in animals receiving PEG-TeNT treatment compared to animals treated with vehicle alone. Bovine cephalic OSA will be observed once weekly and PEG-TeNT-LC-HC doses will be repeated as necessary until efficacy is reduced as determined by recovery of OSA comparable to animals treated with vehicle alone.

Example 20

TeNT epitopes recognized by the major human antibody clonotypes as identified by da Silva antues et al (2017) PloS One,12(1), e0169086 and palero et al (2017) Biotechnology Journal,12(10),1700197 were mapped onto 3-dimensional models of TeNT derived from crystallographic data stored in the protein database (accession ID PDB: 5N0B) using Discovery Studio. Surface serine residues in or around the identified epitope were identified to be mutated to cysteine for subsequent pegylation as described in examples 1-3 and 5.

Example 21

A mixture of PEG-TeNT (5kDa, 10kDa or 20kDa branched or linear PEG attached to surface lysine or cysteine residues) and a 10-1000 fold molar excess of decoy TeNT of 1 nanogram to 64 micrograms was injected intramuscularly into the hind leg muscle of tetanus toxoid vaccinated mice. Local tics greater than the effect observed in mice receiving native TeNT injections will be observed for a period of hours to months.

Example 22

A mixture of 1 nanogram to 64 micrograms of PEG-TeNT (5kDa, 10kDa or 20kDa branched or linear PEG attached to surface lysine or cysteine residues) and a 10-1000 fold molar excess of decoy TeNT was injected intramuscularly into the musculus geniohyoid of humans vaccinated with tetanus toxoid. Local tics greater than the effect observed in humans receiving natural TeNT injections were observed for periods ranging from weeks to months.

Example 23

0.001-10IU/Kg of TeNT is administered intramuscularly to an England bovine head dog having sleep disordered breathing, wherein the dose is administered bilaterally separately to the left and right geniohyoid muscles. After administration, a significant reduction in Obstructive Sleep Apnea (OSA) was observed at the highest dose. The baseline Respiratory Disorder Index (RDI) score was 19.9 (interquartile 5.45) and decreased to 13.2 (interquartile 4.45) after treatment with 10IU/Kg TeNT, which was determined to be significant by the wilson signed rank test compared to placebo administration (P0.043). The bovine dog was observed to maintain a reduction in RDI four months after the trial; the median RDI is 13.4, P is 0.043; the Welch signed rank test.

Sequence listing

<110> Sinogrotox private Co., Ltd

<120> compositions and methods

<130> AU2018902779A

<140> 2018902779

<141> 2018-07-31

<160> 17

<170> PatentIn 3.5 edition

<210> 1

<211> 1314

<212> PRT

<213> Escherichia coli

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Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn Asp

1 5 10 15

Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile Tyr

20 25 30

Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu Arg

35 40 45

Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser Leu

50 55 60

Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr Asp

65 70 75 80

Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn Arg

85 90 95

Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile Asn

100 105 110

Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe Asp

115 120 125

Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro Ser

130 135 140

Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe Gly

145 150 155 160

Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu Arg

165 170 175

Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser Ile

180 185 190

Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn Val

195 200 205

Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe Gln

210 215 220

Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His Gly

225 230 235 240

Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys Gln

245 250 255

Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu Phe

260 265 270

Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys Asn

275 280 285

Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn Lys

290 295 300

Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp Ser

305 310 315 320

Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser Asn

325 330 335

Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn Ser

340 345 350

Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn Ile

355 360 365

Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys Ile

370 375 380

Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe Asn

385 390 395 400

Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met Arg

405 410 415

Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val Ser

420 425 430

Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile Arg

435 440 445

Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu

450 455 460

Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu Lys

465 470 475 480

Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn

485 490 495

Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile

500 505 510

Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr

515 520 525

Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser Asn

530 535 540

Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile Tyr

545 550 555 560

Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile Thr

565 570 575

Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr

580 585 590

Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln Gly

595 600 605

Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn

610 615 620

Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser Thr

625 630 635 640

Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly Tyr

645 650 655

Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu Leu

660 665 670

Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu Ser

675 680 685

Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile Asp

690 695 700

Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys Leu

705 710 715 720

Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys Arg

725 730 735

Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys

740 745 750

Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu

755 760 765

Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu Lys

770 775 780

Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser Ser

785 790 795 800

Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln Leu

805 810 815

Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile Lys

820 825 830

Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser

835 840 845

Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser Lys

850 855 860

Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu

865 870 875 880

Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser

885 890 895

Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln

900 905 910

Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn Glu

915 920 925

Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn Asp

930 935 940

Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val

945 950 955 960

Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile

965 970 975

Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val

980 985 990

Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly

995 1000 1005

Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn

1010 1015 1020

Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp

1025 1030 1035

Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly

1040 1045 1050

Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn

1055 1060 1065

Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val

1070 1075 1080

Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys

1085 1090 1095

Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu

1100 1105 1110

Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr

1115 1120 1125

Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn

1130 1135 1140

Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr Asn

1145 1150 1155

Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys

1160 1165 1170

Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe

1175 1180 1185

Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn

1190 1195 1200

Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn

1205 1210 1215

Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile

1220 1225 1230

Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys

1235 1240 1245

Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser

1250 1255 1260

Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro

1265 1270 1275

Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu

1280 1285 1290

Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp

1295 1300 1305

Glu Gly Trp Thr Asn Asp

1310

<210> 2

<211> 6816

<212> DNA

<213> Escherichia coli

<400> 2

gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 60

agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat 120

catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac 180

gatgacgata aggatcgatg gggatccgag ctcgagccga ttaccatcaa taattttcgt 240

tattcagatc cggtgaataa tgataccatc atcatgatgg aaccaccgta ctgtaaaggg 300

ctggatattt attataaagc gttcaaaatc accgaccgca tctggatcgt gccggaacgc 360

tacgaattcg gcaccaaacc ggaagatttt aatccgccga gtagtctgat cgaaggtgca 420

tcggaatact acgatccgaa ttatctgcgt actgactctg ataaagatcg ctttctgcaa 480

acgatggtaa aactgttcaa tcgtatcaaa aacaatgtag caggcgaagc cctgctggat 540

aaaatcatca acgccattcc gtatctggga aacagttatt ctctgctgga taaattcgat 600

acaaactcga actctgtgtc attcaacctg ctggaacagg acccgagcgg cgcgaccact 660

aagagtgcga tgctgactaa cctgattatt ttcggtccgg gaccggtact gaataaaaat 720

gaagttcgcg gcattgtact gcgtgtcgat aataaaaact atttcccatg tcgtgatggc 780

ttcggcagca tcatgcagat ggccttttgt ccggaatatg tgccaacttt cgataatgtg 840

attgagaaca tcacctctct gacgattggt aaaagtaaat atttccagga tccggctctg 900

ctgctgatgc atgaactgat ccatgttctg catggcctgt atggcatgca ggtttcatcc 960

cacgaaatta tcccatccaa acaggaaatt tacatgcagc atacatatcc gattagtgcc 1020

gaagaactgt tcacttttgg cggccaggat gcgaacctga tttcgattga cattaaaaac 1080

gatctgtatg aaaaaactct gaacgattat aaagcgattg ccaacaaact gtctcaggta 1140

acctcctgta acgatccgaa tattgatatt gacagttata aacaaattta tcagcagaag 1200

tatcagttcg ataaagactc taatggccag tatattgtta acgaagataa attccagatt 1260

ctgtacaata gcattatgta tggctttact gagatcgaac tgggtaaaaa atttaacatc 1320

aagactcgtc tgagctattt tagcatgaat catgatccag tgaaaatccc gaatctgctg 1380

gatgatacga tttataatga taccgaagga tttaacatcg aaagcaagga tctgaaatcc 1440

gaatataaag ggcagaacat gcgcgttaat accaatgcat ttcgcaatgt tgatggttca 1500

ggcctggtgt cgaaactgat tgggctgtgt aagaaaatca ttccaccgac aaatattcgc 1560

gaaaatctgt acaaccgtac ggcgagcctg accgatctgg ggggagaact gtgtattaaa 1620

atcaaaaatg aagatctgac cttcattgct gagaagaata gcttttccga agagccattc 1680

caggacgaaa tcgtgtctta taacaccaag aataaaccgc tgaatttcaa ctactccctg 1740

gacaaaatca ttgtggatta caacctgcag agtaaaatta ccctgccgaa tgatcgtacc 1800

accccggtga cgaaaggcat cccttacgca ccagaatata aatcaaatgc agcctcgact 1860

atcgagatcc ataatattga tgacaacact atttaccagt acctgtatgc tcagaaatct 1920

ccgacgacgc tgcagcgcat caccatgact aacagcgtgg acgatgccct gattaatagc 1980

accaaaatct actcttactt tccgtcggtg atctctaagg ttaatcaggg cgcgcaaggt 2040

atcctgtttc tgcaatgggt gcgtgatatt attgatgatt tcactaatga atctagccag 2100

aaaacgacaa ttgataaaat ttcggatgtt tccaccatcg tgccttacat cggcccagcg 2160

ctgaacatcg tgaagcaggg ttatgagggt aactttatcg gagcactgga aacgaccggc 2220

gtggttctgc tgctggaata tattccggag attactctgc cagttattgc ggctctgtcg 2280

attgcagaga gctcaacgca gaaagaaaaa attattaaga cgatcgacaa tttcctggaa 2340

aagcgctacg aaaaatggat cgaagtgtat aagctggtga aagcgaaatg gctggggacc 2400

gtgaacaccc agttccaaaa acgttcctat caaatgtatc gtagcctgga atatcaggtg 2460

gacgccatta aaaagatcat cgattacgaa tataagatct actccggtcc ggacaaagaa 2520

cagattgcgg acgaaattaa caatctgaaa aataaactgg aggaaaaagc caacaaagcg 2580

atgattaata tcaatatttt catgcgtgaa agcagccgta gcttcctggt caatcagatg 2640

attaatgaag cgaagaaaca actgctggaa tttgatacgc aatctaaaaa tattctgatg 2700

caatacatca aagccaattc taaatttatt gggatcacgg aactgaaaaa gctggaatcg 2760

aaaatcaata aagtctttag caccccgatt ccgttctcct actcgaaaaa tctggattgt 2820

tgggttgaca atgaagaaga tattgatgtt attctgaaaa agagcacgat cctgaacctg 2880

gatattaata acgatattat ctctgatatc agtggtttta attcatcagt tattacttac 2940

ccagacgctc aactggtgcc gggaatcaat gggaaagcca ttcatctggt gaataatgaa 3000

tcaagtgaag tgatcgtgca taaagcgatg gatatcgagt acaacgatat gtttaataat 3060

ttcacggtgt cgttctggct gcgtgttccg aaagtgagtg cctcccacct ggaacaatat 3120

ggaaccaacg aatactcaat cattagcagc atgaagaaac attcgctgag tattggttca 3180

ggttggagcg tttccctgaa agggaacaat ctgatctgga cactgaagga ctcagcgggc 3240

gaagtgcgtc agattacgtt tcgtgatctg ccggataaat ttaatgcata cctggctaac 3300

aaatgggtgt tcatcacaat caccaatgac cgtctgtcgt ctgcaaacct gtatattaat 3360

ggggtactga tgggctcggc agaaattaca gggctgggcg ccattcgtga agataacaat 3420

attacgctga aactggatcg ttgtaataac aataatcagt atgtgagcat tgataaattt 3480

cgtattttct gcaaagcgct gaacccgaaa gaaattgaaa aactgtatac ctcgtatctg 3540

tcaattacgt ttctgcgcga tttctgggga aacccgctgc gttacgatac ggaatactac 3600

ctgatcccgg tagccagttc tagtaaagac gttcaactga aaaatattac cgactacatg 3660

tatctgacaa acgctccatc atacacaaac ggcaaactga acatctatta ccgtcgcctg 3720

tacaatgggc tgaaattcat cattaaacgt tataccccga ataacgaaat tgattccttt 3780

gtgaagtccg gtgacttcat taagctgtat gtatcctata acaataatga acacatcgtt 3840

ggctatccga aggatggcaa tgcctttaac aacctggatc gtattctgcg tgtaggttac 3900

aacgccccgg gtattccgct gtataagaaa atggaagcag tgaaactgcg tgatctgaaa 3960

acatattccg tgcaactgaa gctgtatgat gacaaaaatg ctagcctggg tctggtaggc 4020

acgcataacg gtcagattgg aaacgatcct aatcgtgaca tcctgatcgc ctctaactgg 4080

tattttaacc acctgaaaga taaaattctg ggctgcgatt ggtattttgt ccctaccgat 4140

gaaggctgga cgaacgatta aaagcttgat ccggctgcta acaaagcccg aaaggaagct 4200

gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg 4260

gtcttgaggg gttttttgct gaaaggagga actatatccg gatctggcgt aatagcgaag 4320

aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc 4380

cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 4440

ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 4500

ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 4560

tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 4620

cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 4680

tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 4740

ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 4800

attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg 4860

aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 4920

accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 4980

tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 5040

gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 5100

ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 5160

gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 5220

gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 5280

agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 5340

gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 5400

cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 5460

gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 5520

gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 5580

ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 5640

gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact 5700

ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 5760

tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 5820

actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 5880

taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 5940

gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 6000

tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 6060

ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 6120

gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc 6180

tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 6240

cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 6300

gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 6360

actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 6420

ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 6480

gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 6540

atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 6600

tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 6660

tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 6720

aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 6780

gcctctcccc gcgcgttggc cgattcatta atgcag 6816

<210> 3

<211> 858

<212> PRT

<213> Escherichia coli

<400> 3

Ser Leu Thr Asp Leu Gly Gly Glu Leu Cys Ile Lys Ile Lys Asn Glu

1 5 10 15

Asp Leu Thr Phe Ile Ala Glu Lys Asn Ser Phe Ser Glu Glu Pro Phe

20 25 30

Gln Asp Glu Ile Val Ser Tyr Asn Thr Lys Asn Lys Pro Leu Asn Phe

35 40 45

Asn Tyr Ser Leu Asp Lys Ile Ile Val Asp Tyr Asn Leu Gln Ser Lys

50 55 60

Ile Thr Leu Pro Asn Asp Arg Thr Thr Pro Val Thr Lys Gly Ile Pro

65 70 75 80

Tyr Ala Pro Glu Tyr Lys Ser Asn Ala Ala Ser Thr Ile Glu Ile His

85 90 95

Asn Ile Asp Asp Asn Thr Ile Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser

100 105 110

Pro Thr Thr Leu Gln Arg Ile Thr Met Thr Asn Ser Val Asp Asp Ala

115 120 125

Leu Ile Asn Ser Thr Lys Ile Tyr Ser Tyr Phe Pro Ser Val Ile Ser

130 135 140

Lys Val Asn Gln Gly Ala Gln Gly Ile Leu Phe Leu Gln Trp Val Arg

145 150 155 160

Asp Ile Ile Asp Asp Phe Thr Asn Glu Ser Ser Gln Lys Thr Thr Ile

165 170 175

Asp Lys Ile Ser Asp Val Ser Thr Ile Val Pro Tyr Ile Gly Pro Ala

180 185 190

Leu Asn Ile Val Lys Gln Gly Tyr Glu Gly Asn Phe Ile Gly Ala Leu

195 200 205

Glu Thr Thr Gly Val Val Leu Leu Leu Glu Tyr Ile Pro Glu Ile Thr

210 215 220

Leu Pro Val Ile Ala Ala Leu Ser Ile Ala Glu Ser Ser Thr Gln Lys

225 230 235 240

Glu Lys Ile Ile Lys Thr Ile Asp Asn Phe Leu Glu Lys Arg Tyr Glu

245 250 255

Lys Trp Ile Glu Val Tyr Lys Leu Val Lys Ala Lys Trp Leu Gly Thr

260 265 270

Val Asn Thr Gln Phe Gln Lys Arg Ser Tyr Gln Met Tyr Arg Ser Leu

275 280 285

Glu Tyr Gln Val Asp Ala Ile Lys Lys Ile Ile Asp Tyr Glu Tyr Lys

290 295 300

Ile Tyr Ser Gly Pro Asp Lys Glu Gln Ile Ala Asp Glu Ile Asn Asn

305 310 315 320

Leu Lys Asn Lys Leu Glu Glu Lys Ala Asn Lys Ala Met Ile Asn Ile

325 330 335

Asn Ile Phe Met Arg Glu Ser Ser Arg Ser Phe Leu Val Asn Gln Met

340 345 350

Ile Asn Glu Ala Lys Lys Gln Leu Leu Glu Phe Asp Thr Gln Ser Lys

355 360 365

Asn Ile Leu Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile

370 375 380

Thr Glu Leu Lys Lys Leu Glu Ser Lys Ile Asn Lys Val Phe Ser Thr

385 390 395 400

Pro Ile Pro Phe Ser Tyr Ser Lys Asn Leu Asp Cys Trp Val Asp Asn

405 410 415

Glu Glu Asp Ile Asp Val Ile Leu Lys Lys Ser Thr Ile Leu Asn Leu

420 425 430

Asp Ile Asn Asn Asp Ile Ile Ser Asp Ile Ser Gly Phe Asn Ser Ser

435 440 445

Val Ile Thr Tyr Pro Asp Ala Gln Leu Val Pro Gly Ile Asn Gly Lys

450 455 460

Ala Ile His Leu Val Asn Asn Glu Ser Ser Glu Val Ile Val His Lys

465 470 475 480

Ala Met Asp Ile Glu Tyr Asn Asp Met Phe Asn Asn Phe Thr Val Ser

485 490 495

Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His Leu Glu Gln Tyr

500 505 510

Gly Thr Asn Glu Tyr Ser Ile Ile Ser Ser Met Lys Lys His Ser Leu

515 520 525

Ser Ile Gly Ser Gly Trp Ser Val Ser Leu Lys Gly Asn Asn Leu Ile

530 535 540

Trp Thr Leu Lys Asp Ser Ala Gly Glu Val Arg Gln Ile Thr Phe Arg

545 550 555 560

Asp Leu Pro Asp Lys Phe Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe

565 570 575

Ile Thr Ile Thr Asn Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn

580 585 590

Gly Val Leu Met Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg

595 600 605

Glu Asp Asn Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn

610 615 620

Gln Tyr Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn

625 630 635 640

Pro Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe

645 650 655

Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr

660 665 670

Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn Ile

675 680 685

Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr Asn Gly Lys

690 695 700

Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys Phe Ile Ile

705 710 715 720

Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe Val Lys Ser Gly

725 730 735

Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn Asn Glu His Ile Val

740 745 750

Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn Asn Leu Asp Arg Ile Leu

755 760 765

Arg Val Gly Tyr Asn Ala Pro Gly Ile Pro Leu Tyr Lys Lys Met Glu

770 775 780

Ala Val Lys Leu Arg Asp Leu Lys Thr Tyr Ser Val Gln Leu Lys Leu

785 790 795 800

Tyr Asp Asp Lys Asn Ala Ser Leu Gly Leu Val Gly Thr His Asn Gly

805 810 815

Gln Ile Gly Asn Asp Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp

820 825 830

Tyr Phe Asn His Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe

835 840 845

Val Pro Thr Asp Glu Gly Trp Thr Asn Asp

850 855

<210> 4

<211> 451

<212> PRT

<213> Escherichia coli

<400> 4

Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile

1 5 10 15

Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile

20 25 30

Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala

35 40 45

Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn

50 55 60

Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn

65 70 75 80

Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys

85 90 95

Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile

100 105 110

Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser

115 120 125

Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala

130 135 140

Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn

145 150 155 160

Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp Arg

165 170 175

Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly Ser Ala

180 185 190

Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu

195 200 205

Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys

210 215 220

Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu

225 230 235 240

Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn

245 250 255

Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser Ser

260 265 270

Ser Lys Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr

275 280 285

Asn Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg

290 295 300

Leu Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn

305 310 315 320

Glu Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val

325 330 335

Ser Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn

340 345 350

Ala Phe Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro

355 360 365

Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu

370 375 380

Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser

385 390 395 400

Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro Asn

405 410 415

Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu Lys Asp

420 425 430

Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp

435 440 445

Thr Asn Asp

450

<210> 5

<211> 4234

<212> DNA

<213> Escherichia coli

<400> 5

gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 60

agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat 120

catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac 180

gatgacgata aggatcgatg gggatccgag ctcgagaaaa atctggattg ttgggttgac 240

aatgaagaag atattgatgt tattctgaaa aagagcacga tcctgaacct ggatattaat 300

aacgatatta tctctgatat cagtggtttt aattcatcag ttattactta cccagacgct 360

caactggtgc cgggaatcaa tgggaaagcc attcatctgg tgaataatga atcaagtgaa 420

gtgatcgtgc ataaagcgat ggatatcgag tacaacgata tgtttaataa tttcacggtg 480

tcgttctggc tgcgtgttcc gaaagtgagt gcctcccacc tggaacaata tggaaccaac 540

gaatactcaa tcattagcag catgaagaaa cattcgctga gtattggttc aggttggagc 600

gtttccctga aagggaacaa tctgatctgg acactgaagg actcagcggg cgaagtgcgt 660

cagattacgt ttcgtgatct gccggataaa tttaatgcat acctggctaa caaatgggtg 720

ttcatcacaa tcaccaatga ccgtctgtcg tctgcaaacc tgtatattaa tggggtactg 780

atgggctcgg cagaaattac agggctgggc gccattcgtg aagataacaa tattacgctg 840

aaactggatc gttgtaataa caataatcag tatgtgagca ttgataaatt tcgtattttc 900

tgcaaagcgc tgaacccgaa agaaattgaa aaactgtata cctcgtatct gtcaattacg 960

tttctgcgcg atttctgggg aaacccgctg cgttacgata cggaatacta cctgatcccg 1020

gtagccagtt ctagtaaaga cgttcaactg aaaaatatta ccgactacat gtatctgaca 1080

aacgctccat catacacaaa cggcaaactg aacatctatt accgtcgcct gtacaatggg 1140

ctgaaattca tcattaaacg ttataccccg aataacgaaa ttgattcctt tgtgaagtcc 1200

ggtgacttca ttaagctgta tgtatcctat aacaataatg aacacatcgt tggctatccg 1260

aaggatggca atgcctttaa caacctggat cgtattctgc gtgtaggtta caacgccccg 1320

ggtattccgc tgtataagaa aatggaagca gtgaaactgc gtgatctgaa aacatattcc 1380

gtgcaactga agctgtatga tgacaaaaat gctagcctgg gtctggtagg cacgcataac 1440

ggtcagattg gaaacgatcc taatcgtgac atcctgatcg cctctaactg gtattttaac 1500

cacctgaaag ataaaattct gggctgcgat tggtattttg tccctaccga tgaaggctgg 1560

acgaacgatt aagaattcga agcttgatcc ggctgctaac aaagcccgaa aggaagctga 1620

gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt 1680

cttgaggggt tttttgctga aaggaggaac tatatccgga tctggcgtaa tagcgaagag 1740

gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc 1800

tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 1860

gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 1920

ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 1980

cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc 2040

tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 2100

ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt 2160

ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 2220

tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa 2280

cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 2340

cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 2400

tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 2460

tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 2520

atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 2580

gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 2640

aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 2700

aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 2760

gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 2820

cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 2880

atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 2940

tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 3000

ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 3060

ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 3120

ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 3180

tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 3240

tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 3300

aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 3360

tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 3420

tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 3480

gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 3540

agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 3600

tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 3660

ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 3720

cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 3780

tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 3840

acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 3900

gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 3960

ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 4020

tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 4080

attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 4140

cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc 4200

ctctccccgc gcgttggccg attcattaat gcag 4234

<210> 6

<211> 863

<212> PRT

<213> Escherichia coli

<400> 6

Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn Asp

1 5 10 15

Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile Tyr

20 25 30

Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu Arg

35 40 45

Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser Leu

50 55 60

Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr Asp

65 70 75 80

Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn Arg

85 90 95

Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile Asn

100 105 110

Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe Asp

115 120 125

Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro Ser

130 135 140

Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe Gly

145 150 155 160

Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu Arg

165 170 175

Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser Ile

180 185 190

Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn Val

195 200 205

Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe Gln

210 215 220

Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His Gly

225 230 235 240

Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys Gln

245 250 255

Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu Phe

260 265 270

Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys Asn

275 280 285

Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn Lys

290 295 300

Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp Ser

305 310 315 320

Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser Asn

325 330 335

Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn Ser

340 345 350

Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn Ile

355 360 365

Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys Ile

370 375 380

Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe Asn

385 390 395 400

Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met Arg

405 410 415

Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val Ser

420 425 430

Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile Arg

435 440 445

Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu

450 455 460

Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu Lys

465 470 475 480

Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn

485 490 495

Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile

500 505 510

Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr

515 520 525

Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser Asn

530 535 540

Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile Tyr

545 550 555 560

Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile Thr

565 570 575

Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr

580 585 590

Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln Gly

595 600 605

Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn

610 615 620

Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser Thr

625 630 635 640

Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly Tyr

645 650 655

Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu Leu

660 665 670

Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu Ser

675 680 685

Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile Asp

690 695 700

Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys Leu

705 710 715 720

Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys Arg

725 730 735

Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys

740 745 750

Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu

755 760 765

Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu Lys

770 775 780

Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser Ser

785 790 795 800

Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln Leu

805 810 815

Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile Lys

820 825 830

Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser

835 840 845

Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser

850 855 860

<210> 7

<211> 5479

<212> DNA

<213> Escherichia coli

<400> 7

gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 60

agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat 120

catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac 180

gatgacgata aggatcgatg gggatccgag ctcgagccga ttaccatcaa taattttcgt 240

tattcagatc cggtgaataa tgataccatc atcatgatgg aaccaccgta ctgtaaaggg 300

ctggatattt attataaagc gttcaaaatc accgaccgca tctggatcgt gccggaacgc 360

tacgaattcg gcaccaaacc ggaagatttt aatccgccga gtagtctgat cgaaggtgca 420

tcggaatact acgatccgaa ttatctgcgt actgactctg ataaagatcg ctttctgcaa 480

acgatggtaa aactgttcaa tcgtatcaaa aacaatgtag caggcgaagc cctgctggat 540

aaaatcatca acgccattcc gtatctggga aacagttatt ctctgctgga taaattcgat 600

acaaactcga actctgtgtc attcaacctg ctggaacagg acccgagcgg cgcgaccact 660

aagagtgcga tgctgactaa cctgattatt ttcggtccgg gaccggtact gaataaaaat 720

gaagttcgcg gcattgtact gcgtgtcgat aataaaaact atttcccatg tcgtgatggc 780

ttcggcagca tcatgcagat ggccttttgt ccggaatatg tgccaacttt cgataatgtg 840

attgagaaca tcacctctct gacgattggt aaaagtaaat atttccagga tccggctctg 900

ctgctgatgc atgaactgat ccatgttctg catggcctgt atggcatgca ggtttcatcc 960

cacgaaatta tcccatccaa acaggaaatt tacatgcagc atacatatcc gattagtgcc 1020

gaagaactgt tcacttttgg cggccaggat gcgaacctga tttcgattga cattaaaaac 1080

gatctgtatg aaaaaactct gaacgattat aaagcgattg ccaacaaact gtctcaggta 1140

acctcctgta acgatccgaa tattgatatt gacagttata aacaaattta tcagcagaag 1200

tatcagttcg ataaagactc taatggccag tatattgtta acgaagataa attccagatt 1260

ctgtacaata gcattatgta tggctttact gagatcgaac tgggtaaaaa atttaacatc 1320

aagactcgtc tgagctattt tagcatgaat catgatccag tgaaaatccc gaatctgctg 1380

gatgatacga tttataatga taccgaagga tttaacatcg aaagcaagga tctgaaatcc 1440

gaatataaag ggcagaacat gcgcgttaat accaatgcat ttcgcaatgt tgatggttca 1500

ggcctggtgt cgaaactgat tgggctgtgt aagaaaatca ttccaccgac aaatattcgc 1560

gaaaatctgt acaaccgtac ggcgagcctg accgatctgg ggggagaact gtgtattaaa 1620

atcaaaaatg aagatctgac cttcattgct gagaagaata gcttttccga agagccattc 1680

caggacgaaa tcgtgtctta taacaccaag aataaaccgc tgaatttcaa ctactccctg 1740

gacaaaatca ttgtggatta caacctgcag agtaaaatta ccctgccgaa tgatcgtacc 1800

accccggtga cgaaaggcat cccttacgca ccagaatata aatcaaatgc agcctcgact 1860

atcgagatcc ataatattga tgacaacact atttaccagt acctgtatgc tcagaaatct 1920

ccgacgacgc tgcagcgcat caccatgact aacagcgtgg acgatgccct gattaatagc 1980

accaaaatct actcttactt tccgtcggtg atctctaagg ttaatcaggg cgcgcaaggt 2040

atcctgtttc tgcaatgggt gcgtgatatt attgatgatt tcactaatga atctagccag 2100

aaaacgacaa ttgataaaat ttcggatgtt tccaccatcg tgccttacat cggcccagcg 2160

ctgaacatcg tgaagcaggg ttatgagggt aactttatcg gagcactgga aacgaccggc 2220

gtggttctgc tgctggaata tattccggag attactctgc cagttattgc ggctctgtcg 2280

attgcagaga gctcaacgca gaaagaaaaa attattaaga cgatcgacaa tttcctggaa 2340

aagcgctacg aaaaatggat cgaagtgtat aagctggtga aagcgaaatg gctggggacc 2400

gtgaacaccc agttccaaaa acgttcctat caaatgtatc gtagcctgga atatcaggtg 2460

gacgccatta aaaagatcat cgattacgaa tataagatct actccggtcc ggacaaagaa 2520

cagattgcgg acgaaattaa caatctgaaa aataaactgg aggaaaaagc caacaaagcg 2580

atgattaata tcaatatttt catgcgtgaa agcagccgta gcttcctggt caatcagatg 2640

attaatgaag cgaagaaaca actgctggaa tttgatacgc aatctaaaaa tattctgatg 2700

caatacatca aagccaattc taaatttatt gggatcacgg aactgaaaaa gctggaatcg 2760

aaaatcaata aagtctttag caccccgatt ccgttctcct actcgtaagg taccatggaa 2820

ttcgaagctt gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac 2880

cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt 2940

gctgaaagga ggaactatat ccggatctgg cgtaatagcg aagaggcccg caccgatcgc 3000

ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta 3060

agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 3120

cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 3180

gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 3240

aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 3300

cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 3360

acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 3420

tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta 3480

acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 3540

ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 3600

aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 3660

tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 3720

atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 3780

agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 3840

tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 3900

tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 3960

atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 4020

ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 4080

tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 4140

acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 4200

ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 4260

aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 4320

ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 4380

cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 4440

gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 4500

actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 4560

agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 4620

cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 4680

tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 4740

agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 4800

ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 4860

acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 4920

ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 4980

gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 5040

gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 5100

gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 5160

tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 5220

caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 5280

tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 5340

gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 5400

agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 5460

ggccgattca ttaatgcag 5479

<210> 8

<211> 451

<212> PRT

<213> Escherichia coli

<400> 8

Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile

1 5 10 15

Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile

20 25 30

Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala

35 40 45

Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn

50 55 60

Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn

65 70 75 80

Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys

85 90 95

Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile

100 105 110

Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser

115 120 125

Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala

130 135 140

Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn

145 150 155 160

Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp Arg

165 170 175

Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly Ser Ala

180 185 190

Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu

195 200 205

Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys

210 215 220

Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu

225 230 235 240

Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn

245 250 255

Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser Ser

260 265 270

Ser Lys Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr

275 280 285

Asn Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg

290 295 300

Leu Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn

305 310 315 320

Glu Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val

325 330 335

Ser Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn

340 345 350

Ala Phe Asn Asn Leu Asp Arg Ile Leu Glu Val Gly Tyr Asn Ala Pro

355 360 365

Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu

370 375 380

Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser

385 390 395 400

Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro Asn

405 410 415

Arg Asp Ile Leu Ile Ala Ser Asn Ala Tyr Phe Asn His Leu Lys Asp

420 425 430

Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp

435 440 445

Thr Asn Asp

450

<210> 9

<211> 4234

<212> DNA

<213> Escherichia coli

<400> 9

gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 60

agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat 120

catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac 180

gatgacgata aggatcgatg gggatccgag ctcgagaaaa atttggattg ctgggtcgac 240

aatgaggagg acattgatgt gatattaaag aaatccacta tcttaaatct tgatataaac 300

aatgacatca tctcggatat atctgggttc aattcttccg tcataactta ccctgatgct 360

caactggtac cgggaattaa tgggaaggcc atacacttag tcaacaacga atcttccgag 420

gtgattgtac ataaggcaat ggacatagag tacaatgaca tgtttaataa ctttactgtc 480

tcgttctggt tacgcgtgcc caaagtatct gcctcacacc tggaacagta tggcacaaat 540

gaatattcta ttatcagtag tatgaagaaa cactcgcttt ctataggatc cggctggagt 600

gtttcgctga agggcaataa cttgatctgg acccttaaag attcagcggg agaagtaaga 660

caaataactt tccgggattt gcctgataag ttcaacgcat acctggccaa taagtgggtt 720

ttcataacta taacaaatga ccgcttgtct agcgccaact tatacatcaa tggagtattg 780

atgggttccg ctgagattac aggcttgggt gccataagag aggacaataa tatcaccctg 840

aaactggacc gctgcaataa taacaatcag tacgtgagca tagataaatt ccgtattttt 900

tgcaaagccc tgaacccgaa agaaatcgaa aaactgtata cttcatatct gagcataaca 960

tttctgcgtg atttttgggg taacccgctg cgttatgata ccgaatacta cctgattccg 1020

gttgccagca gcagcaaaga tgttcagctg aaaaatatta ccgactatat gtatctgaca 1080

aatgcgccgt cttataccaa tggcaaactg aatatttatt atcgccggtt gtacaacggg 1140

ctgaagttca ttattaaacg gtacaccccg aacaacgaaa tcgattcatt tgttaaatcc 1200

ggggatttca taaagttata cgtgagctat aataacaacg agcatattgt aggttatccg 1260

aaagacggta atgctttcaa caacttggat cggatactgg aagttggtta caacgcccca 1320

ggtattccac tgtataagaa aatggaagcc gtcaagttgc gtgatttaaa gacgtactca 1380

gtacagctta aattatacga cgataagaat gcaagccttg gattggttgg gacccacaat 1440

ggtcagattg gaaatgaccc caatcgggac attctgatag catccaacgc gtacttcaac 1500

catttgaaag ataaaatcct gggctgtgat tggtactttg taccgactga tgaaggatgg 1560

acgaatgact aagaattcga agcttgatcc ggctgctaac aaagcccgaa aggaagctga 1620

gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt 1680

cttgaggggt tttttgctga aaggaggaac tatatccgga tctggcgtaa tagcgaagag 1740

gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc 1800

tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 1860

gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 1920

ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 1980

cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc 2040

tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 2100

ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt 2160

ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 2220

tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa 2280

cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 2340

cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 2400

tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 2460

tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 2520

atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 2580

gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 2640

aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 2700

aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 2760

gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 2820

cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 2880

atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 2940

tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 3000

ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 3060

ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 3120

ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 3180

tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 3240

tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 3300

aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 3360

tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 3420

tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 3480

gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 3540

agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 3600

tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 3660

ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 3720

cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 3780

tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 3840

acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 3900

gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 3960

ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 4020

tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 4080

attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 4140

cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc 4200

ctctccccgc gcgttggccg attcattaat gcag 4234

<210> 10

<211> 1314

<212> PRT

<213> Escherichia coli

<400> 10

Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn Asp

1 5 10 15

Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile Tyr

20 25 30

Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu Arg

35 40 45

Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser Leu

50 55 60

Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr Asp

65 70 75 80

Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn Arg

85 90 95

Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile Asn

100 105 110

Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe Asp

115 120 125

Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro Ser

130 135 140

Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe Gly

145 150 155 160

Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu Arg

165 170 175

Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser Ile

180 185 190

Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn Val

195 200 205

Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe Gln

210 215 220

Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His Gly

225 230 235 240

Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys Gln

245 250 255

Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu Phe

260 265 270

Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys Asn

275 280 285

Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn Lys

290 295 300

Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp Ser

305 310 315 320

Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser Asn

325 330 335

Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn Ser

340 345 350

Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn Ile

355 360 365

Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys Ile

370 375 380

Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe Asn

385 390 395 400

Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met Arg

405 410 415

Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val Ser

420 425 430

Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile Arg

435 440 445

Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu

450 455 460

Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu Lys

465 470 475 480

Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn

485 490 495

Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile

500 505 510

Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr

515 520 525

Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser Asn

530 535 540

Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile Tyr

545 550 555 560

Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile Thr

565 570 575

Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr

580 585 590

Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln Gly

595 600 605

Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn

610 615 620

Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser Thr

625 630 635 640

Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly Tyr

645 650 655

Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu Leu

660 665 670

Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu Ser

675 680 685

Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile Asp

690 695 700

Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys Leu

705 710 715 720

Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys Arg

725 730 735

Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys

740 745 750

Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu

755 760 765

Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu Lys

770 775 780

Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser Ser

785 790 795 800

Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln Leu

805 810 815

Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile Lys

820 825 830

Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser

835 840 845

Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser Lys

850 855 860

Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu

865 870 875 880

Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser

885 890 895

Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln

900 905 910

Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn Glu

915 920 925

Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn Asp

930 935 940

Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val

945 950 955 960

Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile

965 970 975

Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val

980 985 990

Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly

995 1000 1005

Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn

1010 1015 1020

Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp

1025 1030 1035

Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly

1040 1045 1050

Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn

1055 1060 1065

Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val

1070 1075 1080

Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys

1085 1090 1095

Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu

1100 1105 1110

Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr

1115 1120 1125

Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn

1130 1135 1140

Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr Asn

1145 1150 1155

Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys

1160 1165 1170

Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe

1175 1180 1185

Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn

1190 1195 1200

Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn

1205 1210 1215

Asn Leu Asp Arg Ile Leu Glu Val Gly Tyr Asn Ala Pro Gly Ile

1220 1225 1230

Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys

1235 1240 1245

Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser

1250 1255 1260

Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro

1265 1270 1275

Asn Arg Asp Ile Leu Ile Ala Ser Asn Ala Tyr Phe Asn His Leu

1280 1285 1290

Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp

1295 1300 1305

Glu Gly Trp Thr Asn Asp

1310

<210> 11

<211> 6816

<212> DNA

<213> Escherichia coli

<400> 11

gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 60

agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat 120

catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac 180

gatgacgata aggatcgatg gggatccgag ctcgagccga tcaccatcaa caacttccgt 240

tactctgacc cggttaacaa cgacaccatc atcatgatgg aaccgccgta ctgcaaaggt 300

ctggacatct actacaaagc gttcaaaatc accgaccgta tctggatcgt tccggaacgt 360

tacgaattcg gtaccaaacc ggaagacttc aacccgccgt cttctctgat cgaaggtgcg 420

tctgaatact acgacccgaa ctacctgcgt accgactctg acaaagaccg tttcctgcag 480

accatggtta aactgttcaa ccgtatcaaa aacaacgttg cgggtgaagc gctgctggac 540

aaaatcatca acgcgatccc gtacctgggt aactcttact ctctgctgga caaattcgac 600

accaactcta actctgtttc tttcaacctg ctggaacagg acccgtctgg tgcgaccacc 660

aaatctgcga tgctgaccaa cctgatcatc ttcggtccgg gtccggttct gaacaaaaac 720

gaagttcgtg gtatcgttct gcgtgttgac aacaaaaact acttcccgtg ccgtgacggt 780

ttcggttcta tcatgcagat ggcgttctgc ccggaatacg ttccgacctt cgacaacgtt 840

atcgaaaaca tcacctctct gaccatcggt aaatctaaat acttccagga cccggcgctg 900

ctgctgatgc acgaactgat ccacgttctg cacggtctgt acggtatgca ggtttcttct 960

cacgaaatca tcccgtctaa acaggaaatc tacatgcagc acacctaccc gatctctgcg 1020

gaagaactgt tcaccttcgg tggtcaggac gcgaacctga tctctatcga catcaaaaac 1080

gacctgtacg aaaaaaccct gaacgactac aaagcgatcg cgaacaaact gtctcaggtt 1140

acctcttgca acgacccgaa catcgacatc gactcttaca aacagatcta ccagcagaaa 1200

taccagttcg acaaagactc taacggtcag tacatcgtta acgaagacaa attccagatc 1260

ctgtacaact ctatcatgta cggtttcacc gaaatcgaac tgggtaaaaa attcaacatc 1320

aaaacccgtc tgtcttactt ctctatgaac cacgacccgg ttaaaatccc gaacctgctg 1380

gacgacacca tctacaacga caccgaaggt ttcaacatcg aatctaaaga cctgaaatct 1440

gaatacaaag gtcagaacat gcgtgttaac accaacgcgt tccgtaacgt tgacggttct 1500

ggtctggttt ctaaactgat cggtctgtgc aaaaaaatca tcccgccgac caacatccgt 1560

gaaaacctgt acaaccgtac cgcgtctctg accgacctgg gtggtgaact gtgcatcaaa 1620

atcaaaaacg aagacctgac cttcatcgcg gaaaaaaact ctttctctga agaaccgttc 1680

caggacgaaa tcgtttctta caacaccaaa aacaaaccgc tgaacttcaa ctactctctg 1740

gacaaaatca tcgttgacta caacctgcag tctaaaatca ccctgccgaa cgaccgtacc 1800

accccggtta ccaaaggtat cccgtacgcg ccggaataca aatctaacgc ggcgtctacc 1860

atcgaaatcc acaacatcga cgacaacacc atctaccagt acctgtacgc gcagaaatct 1920

ccgaccaccc tgcagcgtat caccatgacc aactctgttg acgacgcgct gatcaactct 1980

accaaaatct actcttactt cccgtctgtt atctctaaag ttaaccaggg tgcgcagggt 2040

atcctgttcc tgcagtgggt tcgtgacatc atcgacgact tcaccaacga atcttctcag 2100

aaaaccacca tcgacaaaat ctctgacgtt tctaccatcg ttccgtacat cggtccggcg 2160

ctgaacatcg ttaaacaggg ttacgaaggt aacttcatcg gtgcgctgga aaccaccggt 2220

gttgttctgc tgctggaata catcccggaa atcaccctgc cggttatcgc ggcgctgtct 2280

atcgcggaat cttctaccca gaaagaaaaa atcatcaaaa ccatcgacaa cttcctggaa 2340

aaacgttacg aaaaatggat cgaagtttac aaactggtta aagcgaaatg gctgggtacc 2400

gttaacaccc agttccagaa acgttcttac cagatgtacc gttctctgga ataccaggtt 2460

gacgcgatca aaaaaatcat cgactacgaa tacaaaatct actctggtcc ggacaaagaa 2520

cagatcgcgg acgaaatcaa caacctgaaa aacaaactgg aagaaaaagc gaacaaagcg 2580

atgatcaaca tcaacatctt catgcgtgaa tcttctcgtt ctttcctggt taaccagatg 2640

atcaacgaag cgaaaaaaca gctgctggaa ttcgacaccc agtctaaaaa catcctgatg 2700

cagtacatca aagcgaactc taaattcatc ggtatcaccg aactgaaaaa actggaatct 2760

aaaatcaaca aagttttctc taccccgatc ccgttctctt actctaaaaa cctggactgc 2820

tgggttgaca acgaagaaga catcgacgtt atcctgaaaa aatctaccat cctgaacctg 2880

gacatcaaca acgacatcat ctctgacatc tctggtttca actcttctgt tatcacctac 2940

ccggacgcgc agctggttcc gggtatcaac ggtaaagcga tccacctggt taacaacgaa 3000

tcttctgaag ttatcgttca caaagcgatg gacatcgaat acaacgacat gttcaacaac 3060

ttcaccgttt ctttctggct gcgtgttccg aaagtttctg cgtctcacct ggaacagtac 3120

ggtaccaacg aatactctat catctcttct atgaaaaaac actctctgtc tatcggttct 3180

ggttggtctg tttctctgaa aggtaacaac ctgatctgga ccctgaaaga ctctgcgggt 3240

gaagttcgtc agatcacctt ccgtgacctg ccggacaaat tcaacgcgta cctggcgaac 3300

aaatgggttt tcatcaccat caccaacgac cgtctgtctt ctgcgaacct gtacatcaac 3360

ggtgttctga tgggttctgc ggaaatcacc ggtctgggtg cgatccgtga agacaacaac 3420

atcaccctga aactggaccg ttgcaacaac aacaaccagt acgtttctat cgacaaattc 3480

cgtatcttct gcaaagcgct gaacccgaaa gaaatcgaaa aactgtacac ctcttacctg 3540

tctatcacct tcctgcgtga cttctggggt aacccgctgc gttacgacac cgaatactac 3600

ctgatcccgg ttgcgtcttc ttctaaagac gttcagctga aaaacatcac cgactacatg 3660

tacctgacca acgcgccgtc ttacaccaac ggtaaactga acatctacta ccgtcgtctg 3720

tacaacggtc tgaaattcat catcaaacgt tacaccccga acaacgaaat cgactctttc 3780

gttaaatctg gtgacttcat caaactgtac gtttcttaca acaacaacga acacatcgtt 3840

ggttacccga aagacggtaa cgcgttcaac aacctggacc gtatcctgga agttggttac 3900

aacgcgccgg gtatcccgct gtacaaaaaa atggaagcgg ttaaactgcg tgacctgaaa 3960

acctactctg ttcagctgaa actgtacgac gacaaaaacg cgtctctggg tctggttggt 4020

acccacaacg gtcagatcgg taacgacccg aaccgtgaca tcctgatcgc gtctaacgcg 4080

tacttcaacc acctgaaaga caaaatcctg ggttgcgact ggtacttcgt tccgaccgac 4140

gaaggttgga ccaacgacta aaagcttgat ccggctgcta acaaagcccg aaaggaagct 4200

gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg 4260

gtcttgaggg gttttttgct gaaaggagga actatatccg gatctggcgt aatagcgaag 4320

aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc 4380

cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 4440

ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 4500

ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 4560

tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 4620

cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 4680

tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 4740

ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 4800

attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg 4860

aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 4920

accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 4980

tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 5040

gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 5100

ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 5160

gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 5220

gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 5280

agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 5340

gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 5400

cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 5460

gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 5520

gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 5580

ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 5640

gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact 5700

ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 5760

tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 5820

actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 5880

taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 5940

gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 6000

tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 6060

ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 6120

gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc 6180

tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 6240

cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 6300

gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 6360

actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 6420

ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 6480

gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 6540

atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 6600

tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 6660

tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 6720

aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 6780

gcctctcccc gcgcgttggc cgattcatta atgcag 6816

<210> 12

<211> 1314

<212> PRT

<213> Escherichia coli

<400> 12

Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn Asp

1 5 10 15

Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile Tyr

20 25 30

Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu Arg

35 40 45

Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser Leu

50 55 60

Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr Asp

65 70 75 80

Cys Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn Arg

85 90 95

Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile Asn

100 105 110

Ala Ile Pro Tyr Leu Gly Asn Cys Tyr Ser Leu Leu Asp Lys Phe Asp

115 120 125

Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro Cys

130 135 140

Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe Gly

145 150 155 160

Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu Arg

165 170 175

Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser Ile

180 185 190

Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn Val

195 200 205

Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe Gln

210 215 220

Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His Gly

225 230 235 240

Leu Tyr Gly Met Gln Val Ser Cys His Glu Ile Ile Pro Ser Lys Gln

245 250 255

Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu Phe

260 265 270

Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys Asn

275 280 285

Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn Lys

290 295 300

Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp Ser

305 310 315 320

Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Cys Asn

325 330 335

Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn Ser

340 345 350

Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn Ile

355 360 365

Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys Ile

370 375 380

Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe Asn

385 390 395 400

Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met Arg

405 410 415

Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Cys Gly Leu Val Ser

420 425 430

Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile Arg

435 440 445

Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu

450 455 460

Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu Lys

465 470 475 480

Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn

485 490 495

Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile

500 505 510

Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr

515 520 525

Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser Asn

530 535 540

Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile Tyr

545 550 555 560

Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile Thr

565 570 575

Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr

580 585 590

Ser Tyr Phe Pro Ser Val Ile Cys Lys Val Asn Gln Gly Ala Gln Gly

595 600 605

Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn

610 615 620

Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser Thr

625 630 635 640

Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly Tyr

645 650 655

Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu Leu

660 665 670

Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu Ser

675 680 685

Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile Asp

690 695 700

Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys Leu

705 710 715 720

Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys Arg

725 730 735

Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys

740 745 750

Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu

755 760 765

Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu Lys

770 775 780

Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser Ser

785 790 795 800

Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln Leu

805 810 815

Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile Lys

820 825 830

Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser

835 840 845

Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser Lys

850 855 860

Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu

865 870 875 880

Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser

885 890 895

Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln

900 905 910

Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn Glu

915 920 925

Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn Asp

930 935 940

Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val

945 950 955 960

Ser Ala Cys His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile

965 970 975

Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val

980 985 990

Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly

995 1000 1005

Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn

1010 1015 1020

Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp

1025 1030 1035

Arg Leu Cys Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly

1040 1045 1050

Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn

1055 1060 1065

Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val

1070 1075 1080

Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys

1085 1090 1095

Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu

1100 1105 1110

Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr

1115 1120 1125

Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn

1130 1135 1140

Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Cys Tyr Thr Asn

1145 1150 1155

Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys

1160 1165 1170

Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Cys Phe

1175 1180 1185

Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn

1190 1195 1200

Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn

1205 1210 1215

Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile

1220 1225 1230

Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys

1235 1240 1245

Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser

1250 1255 1260

Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro

1265 1270 1275

Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu

1280 1285 1290

Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp

1295 1300 1305

Glu Gly Trp Thr Asn Asp

1310

<210> 13

<211> 6816

<212> DNA

<213> Escherichia coli

<400> 13

gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 60

agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat 120

catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac 180

gatgacgata aggatcgatg gggatccgag ctcgagccga tcaccatcaa caacttccgt 240

tactctgacc cggttaacaa cgacaccatc atcatgatgg aaccgccgta ctgcaaaggt 300

ctggacatct actacaaagc gttcaaaatc accgaccgta tctggatcgt tccggaacgt 360

tacgaattcg gtaccaaacc ggaagacttc aacccgccgt cttctctgat cgaaggtgcg 420

tctgaatact acgacccgaa ctacctgcgt accgactgcg acaaagaccg tttcctgcag 480

accatggtta aactgttcaa ccgtatcaaa aacaacgttg cgggtgaagc gctgctggac 540

aaaatcatca acgcgatccc gtacctgggt aactgctact ctctgctgga caaattcgac 600

accaactcta actctgtttc tttcaacctg ctggaacagg acccgtgcgg tgcgaccacc 660

aaatctgcga tgctgaccaa cctgatcatc ttcggtccgg gtccggttct gaacaaaaac 720

gaagttcgtg gtatcgttct gcgtgttgac aacaaaaact acttcccgtg ccgtgacggt 780

ttcggttcta tcatgcagat ggcgttctgc ccggaatacg ttccgacctt cgacaacgtt 840

atcgaaaaca tcacctctct gaccatcggt aaatctaaat acttccagga cccggcgctg 900

ctgctgatgc acgaactgat ccacgttctg cacggtctgt acggtatgca ggtttcttgc 960

cacgaaatca tcccgtctaa acaggaaatc tacatgcagc acacctaccc gatctctgcg 1020

gaagaactgt tcaccttcgg tggtcaggac gcgaacctga tctctatcga catcaaaaac 1080

gacctgtacg aaaaaaccct gaacgactac aaagcgatcg cgaacaaact gtctcaggtt 1140

acctcttgca acgacccgaa catcgacatc gactcttaca aacagatcta ccagcagaaa 1200

taccagttcg acaaagactg caacggtcag tacatcgtta acgaagacaa attccagatc 1260

ctgtacaact ctatcatgta cggtttcacc gaaatcgaac tgggtaaaaa attcaacatc 1320

aaaacccgtc tgtcttactt ctctatgaac cacgacccgg ttaaaatccc gaacctgctg 1380

gacgacacca tctacaacga caccgaaggt ttcaacatcg aatctaaaga cctgaaatct 1440

gaatacaaag gtcagaacat gcgtgttaac accaacgcgt tccgtaacgt tgacggttgc 1500

ggtctggttt ctaaactgat cggtctgtgc aaaaaaatca tcccgccgac caacatccgt 1560

gaaaacctgt acaaccgtac cgcgtctctg accgacctgg gtggtgaact gtgcatcaaa 1620

atcaaaaacg aagacctgac cttcatcgcg gaaaaaaact ctttctctga agaaccgttc 1680

caggacgaaa tcgtttctta caacaccaaa aacaaaccgc tgaacttcaa ctactctctg 1740

gacaaaatca tcgttgacta caacctgcag tctaaaatca ccctgccgaa cgaccgtacc 1800

accccggtta ccaaaggtat cccgtacgcg ccggaataca aatctaacgc ggcgtctacc 1860

atcgaaatcc acaacatcga cgacaacacc atctaccagt acctgtacgc gcagaaatct 1920

ccgaccaccc tgcagcgtat caccatgacc aactctgttg acgacgcgct gatcaactct 1980

accaaaatct actcttactt cccgtctgtt atctgcaaag ttaaccaggg tgcgcagggt 2040

atcctgttcc tgcagtgggt tcgtgacatc atcgacgact tcaccaacga atcttctcag 2100

aaaaccacca tcgacaaaat ctctgacgtt tctaccatcg ttccgtacat cggtccggcg 2160

ctgaacatcg ttaaacaggg ttacgaaggt aacttcatcg gtgcgctgga aaccaccggt 2220

gttgttctgc tgctggaata catcccggaa atcaccctgc cggttatcgc ggcgctgtct 2280

atcgcggaat cttctaccca gaaagaaaaa atcatcaaaa ccatcgacaa cttcctggaa 2340

aaacgttacg aaaaatggat cgaagtttac aaactggtta aagcgaaatg gctgggtacc 2400

gttaacaccc agttccagaa acgttcttac cagatgtacc gttctctgga ataccaggtt 2460

gacgcgatca aaaaaatcat cgactacgaa tacaaaatct actctggtcc ggacaaagaa 2520

cagatcgcgg acgaaatcaa caacctgaaa aacaaactgg aagaaaaagc gaacaaagcg 2580

atgatcaaca tcaacatctt catgcgtgaa tcttctcgtt ctttcctggt taaccagatg 2640

atcaacgaag cgaaaaaaca gctgctggaa ttcgacaccc agtctaaaaa catcctgatg 2700

cagtacatca aagcgaactc taaattcatc ggtatcaccg aactgaaaaa actggaatct 2760

aaaatcaaca aagttttctc taccccgatc ccgttctctt actctaaaaa cctggactgc 2820

tgggttgaca acgaagaaga catcgacgtt atcctgaaaa aatctaccat cctgaacctg 2880

gacatcaaca acgacatcat ctctgacatc tctggtttca actcttctgt tatcacctac 2940

ccggacgcgc agctggttcc gggtatcaac ggtaaagcga tccacctggt taacaacgaa 3000

tcttctgaag ttatcgttca caaagcgatg gacatcgaat acaacgacat gttcaacaac 3060

ttcaccgttt ctttctggct gcgtgttccg aaagtttctg cgtgccacct ggaacagtac 3120

ggtaccaacg aatactctat catctcttct atgaaaaaac actctctgtc tatcggttct 3180

ggttggtctg tttctctgaa aggtaacaac ctgatctgga ccctgaaaga ctctgcgggt 3240

gaagttcgtc agatcacctt ccgtgacctg ccggacaaat tcaacgcgta cctggcgaac 3300

aaatgggttt tcatcaccat caccaacgac cgtctgtgct ctgcgaacct gtacatcaac 3360

ggtgttctga tgggttctgc ggaaatcacc ggtctgggtg cgatccgtga agacaacaac 3420

atcaccctga aactggaccg ttgcaacaac aacaaccagt acgtttctat cgacaaattc 3480

cgtatcttct gcaaagcgct gaacccgaaa gaaatcgaaa aactgtacac ctcttacctg 3540

tctatcacct tcctgcgtga cttctggggt aacccgctgc gttacgacac cgaatactac 3600

ctgatcccgg ttgcgtcttc ttctaaagac gttcagctga aaaacatcac cgactacatg 3660

tacctgacca acgcgccgtg ctacaccaac ggtaaactga acatctacta ccgtcgtctg 3720

tacaacggtc tgaaattcat catcaaacgt tacaccccga acaacgaaat cgactgcttc 3780

gttaaatctg gtgacttcat caaactgtac gtttcttaca acaacaacga acacatcgtt 3840

ggttacccga aagacggtaa cgcgttcaac aacctggacc gtatcctgcg tgttggttac 3900

aacgcgccgg gtatcccgct gtacaaaaaa atggaagcgg ttaaactgcg tgacctgaaa 3960

acctactctg ttcagctgaa actgtacgac gacaaaaacg cgtctctggg tctggttggt 4020

acccacaacg gtcagatcgg taacgacccg aaccgtgaca tcctgatcgc gtctaactgg 4080

tacttcaacc acctgaaaga caaaatcctg ggttgcgact ggtacttcgt tccgaccgac 4140

gaaggttgga ccaacgacta aaagcttgat ccggctgcta acaaagcccg aaaggaagct 4200

gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg 4260

gtcttgaggg gttttttgct gaaaggagga actatatccg gatctggcgt aatagcgaag 4320

aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc 4380

cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 4440

ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 4500

ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 4560

tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 4620

cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 4680

tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 4740

ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 4800

attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg 4860

aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 4920

accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 4980

tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 5040

gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 5100

ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 5160

gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 5220

gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 5280

agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 5340

gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 5400

cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 5460

gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 5520

gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 5580

ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 5640

gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact 5700

ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 5760

tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 5820

actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 5880

taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 5940

gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 6000

tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 6060

ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 6120

gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc 6180

tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 6240

cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 6300

gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 6360

actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 6420

ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 6480

gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 6540

atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 6600

tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 6660

tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 6720

aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 6780

gcctctcccc gcgcgttggc cgattcatta atgcag 6816

<210> 14

<211> 1314

<212> PRT

<213> Escherichia coli

<400> 14

Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn Asp

1 5 10 15

Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile Tyr

20 25 30

Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu Arg

35 40 45

Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser Leu

50 55 60

Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr Asp

65 70 75 80

Cys Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn Arg

85 90 95

Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile Asn

100 105 110

Ala Ile Pro Tyr Leu Gly Asn Cys Tyr Ser Leu Leu Asp Lys Phe Asp

115 120 125

Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro Cys

130 135 140

Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe Gly

145 150 155 160

Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu Arg

165 170 175

Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser Ile

180 185 190

Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn Val

195 200 205

Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe Gln

210 215 220

Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His Gly

225 230 235 240

Leu Tyr Gly Met Gln Val Ser Cys His Glu Ile Ile Pro Ser Lys Gln

245 250 255

Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu Phe

260 265 270

Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys Asn

275 280 285

Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn Lys

290 295 300

Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp Ser

305 310 315 320

Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Cys Asn

325 330 335

Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn Ser

340 345 350

Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn Ile

355 360 365

Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys Ile

370 375 380

Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe Asn

385 390 395 400

Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met Arg

405 410 415

Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Cys Gly Leu Val Ser

420 425 430

Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile Arg

435 440 445

Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu

450 455 460

Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu Lys

465 470 475 480

Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn

485 490 495

Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile

500 505 510

Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr

515 520 525

Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser Asn

530 535 540

Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile Tyr

545 550 555 560

Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile Thr

565 570 575

Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr

580 585 590

Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln Gly

595 600 605

Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn

610 615 620

Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser Thr

625 630 635 640

Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly Tyr

645 650 655

Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu Leu

660 665 670

Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu Ser

675 680 685

Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile Asp

690 695 700

Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys Leu

705 710 715 720

Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys Arg

725 730 735

Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys

740 745 750

Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu

755 760 765

Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu Lys

770 775 780

Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser Ser

785 790 795 800

Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln Leu

805 810 815

Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile Lys

820 825 830

Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser

835 840 845

Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser Lys

850 855 860

Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu

865 870 875 880

Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser

885 890 895

Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln

900 905 910

Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn Glu

915 920 925

Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn Asp

930 935 940

Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val

945 950 955 960

Ser Ala Cys His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile

965 970 975

Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val

980 985 990

Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly

995 1000 1005

Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn

1010 1015 1020

Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp

1025 1030 1035

Arg Leu Cys Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly

1040 1045 1050

Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn

1055 1060 1065

Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val

1070 1075 1080

Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys

1085 1090 1095

Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu

1100 1105 1110

Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr

1115 1120 1125

Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn

1130 1135 1140

Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Cys Tyr Thr Asn

1145 1150 1155

Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys

1160 1165 1170

Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Cys Phe

1175 1180 1185

Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn

1190 1195 1200

Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn

1205 1210 1215

Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile

1220 1225 1230

Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys

1235 1240 1245

Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser

1250 1255 1260

Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro

1265 1270 1275

Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu

1280 1285 1290

Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp

1295 1300 1305

Glu Gly Trp Thr Asn Asp

1310

<210> 15

<211> 6816

<212> DNA

<213> Escherichia coli

<400> 15

gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 60

agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat 120

catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac 180

gatgacgata aggatcgatg gggatccgag ctcgagccga tcaccatcaa caacttccgt 240

tactctgacc cggttaacaa cgacaccatc atcatgatgg aaccgccgta ctgcaaaggt 300

ctggacatct actacaaagc gttcaaaatc accgaccgta tctggatcgt tccggaacgt 360

tacgaattcg gtaccaaacc ggaagacttc aacccgccgt cttctctgat cgaaggtgcg 420

tctgaatact acgacccgaa ctacctgcgt accgactgcg acaaagaccg tttcctgcag 480

accatggtta aactgttcaa ccgtatcaaa aacaacgttg cgggtgaagc gctgctggac 540

aaaatcatca acgcgatccc gtacctgggt aactgctact ctctgctgga caaattcgac 600

accaactcta actctgtttc tttcaacctg ctggaacagg acccgtgcgg tgcgaccacc 660

aaatctgcga tgctgaccaa cctgatcatc ttcggtccgg gtccggttct gaacaaaaac 720

gaagttcgtg gtatcgttct gcgtgttgac aacaaaaact acttcccgtg ccgtgacggt 780

ttcggttcta tcatgcagat ggcgttctgc ccggaatacg ttccgacctt cgacaacgtt 840

atcgaaaaca tcacctctct gaccatcggt aaatctaaat acttccagga cccggcgctg 900

ctgctgatgc acgaactgat ccacgttctg cacggtctgt acggtatgca ggtttcttgc 960

cacgaaatca tcccgtctaa acaggaaatc tacatgcagc acacctaccc gatctctgcg 1020

gaagaactgt tcaccttcgg tggtcaggac gcgaacctga tctctatcga catcaaaaac 1080

gacctgtacg aaaaaaccct gaacgactac aaagcgatcg cgaacaaact gtctcaggtt 1140

acctcttgca acgacccgaa catcgacatc gactcttaca aacagatcta ccagcagaaa 1200

taccagttcg acaaagactg caacggtcag tacatcgtta acgaagacaa attccagatc 1260

ctgtacaact ctatcatgta cggtttcacc gaaatcgaac tgggtaaaaa attcaacatc 1320

aaaacccgtc tgtcttactt ctctatgaac cacgacccgg ttaaaatccc gaacctgctg 1380

gacgacacca tctacaacga caccgaaggt ttcaacatcg aatctaaaga cctgaaatct 1440

gaatacaaag gtcagaacat gcgtgttaac accaacgcgt tccgtaacgt tgacggttgc 1500

ggtctggttt ctaaactgat cggtctgtgc aaaaaaatca tcccgccgac caacatccgt 1560

gaaaacctgt acaaccgtac cgcgtctctg accgacctgg gtggtgaact gtgcatcaaa 1620

atcaaaaacg aagacctgac cttcatcgcg gaaaaaaact ctttctctga agaaccgttc 1680

caggacgaaa tcgtttctta caacaccaaa aacaaaccgc tgaacttcaa ctactctctg 1740

gacaaaatca tcgttgacta caacctgcag tctaaaatca ccctgccgaa cgaccgtacc 1800

accccggtta ccaaaggtat cccgtacgcg ccggaataca aatctaacgc ggcgtctacc 1860

atcgaaatcc acaacatcga cgacaacacc atctaccagt acctgtacgc gcagaaatct 1920

ccgaccaccc tgcagcgtat caccatgacc aactctgttg acgacgcgct gatcaactct 1980

accaaaatct actcttactt cccgtctgtt atctctaaag ttaaccaggg tgcgcagggt 2040

atcctgttcc tgcagtgggt tcgtgacatc atcgacgact tcaccaacga atcttctcag 2100

aaaaccacca tcgacaaaat ctctgacgtt tctaccatcg ttccgtacat cggtccggcg 2160

ctgaacatcg ttaaacaggg ttacgaaggt aacttcatcg gtgcgctgga aaccaccggt 2220

gttgttctgc tgctggaata catcccggaa atcaccctgc cggttatcgc ggcgctgtct 2280

atcgcggaat cttctaccca gaaagaaaaa atcatcaaaa ccatcgacaa cttcctggaa 2340

aaacgttacg aaaaatggat cgaagtttac aaactggtta aagcgaaatg gctgggtacc 2400

gttaacaccc agttccagaa acgttcttac cagatgtacc gttctctgga ataccaggtt 2460

gacgcgatca aaaaaatcat cgactacgaa tacaaaatct actctggtcc ggacaaagaa 2520

cagatcgcgg acgaaatcaa caacctgaaa aacaaactgg aagaaaaagc gaacaaagcg 2580

atgatcaaca tcaacatctt catgcgtgaa tcttctcgtt ctttcctggt taaccagatg 2640

atcaacgaag cgaaaaaaca gctgctggaa ttcgacaccc agtctaaaaa catcctgatg 2700

cagtacatca aagcgaactc taaattcatc ggtatcaccg aactgaaaaa actggaatct 2760

aaaatcaaca aagttttctc taccccgatc ccgttctctt actctaaaaa cctggactgc 2820

tgggttgaca acgaagaaga catcgacgtt atcctgaaaa aatctaccat cctgaacctg 2880

gacatcaaca acgacatcat ctctgacatc tctggtttca actcttctgt tatcacctac 2940

ccggacgcgc agctggttcc gggtatcaac ggtaaagcga tccacctggt taacaacgaa 3000

tcttctgaag ttatcgttca caaagcgatg gacatcgaat acaacgacat gttcaacaac 3060

ttcaccgttt ctttctggct gcgtgttccg aaagtttctg cgtgccacct ggaacagtac 3120

ggtaccaacg aatactctat catctcttct atgaaaaaac actctctgtc tatcggttct 3180

ggttggtctg tttctctgaa aggtaacaac ctgatctgga ccctgaaaga ctctgcgggt 3240

gaagttcgtc agatcacctt ccgtgacctg ccggacaaat tcaacgcgta cctggcgaac 3300

aaatgggttt tcatcaccat caccaacgac cgtctgtgct ctgcgaacct gtacatcaac 3360

ggtgttctga tgggttctgc ggaaatcacc ggtctgggtg cgatccgtga agacaacaac 3420

atcaccctga aactggaccg ttgcaacaac aacaaccagt acgtttctat cgacaaattc 3480

cgtatcttct gcaaagcgct gaacccgaaa gaaatcgaaa aactgtacac ctcttacctg 3540

tctatcacct tcctgcgtga cttctggggt aacccgctgc gttacgacac cgaatactac 3600

ctgatcccgg ttgcgtcttc ttctaaagac gttcagctga aaaacatcac cgactacatg 3660

tacctgacca acgcgccgtg ctacaccaac ggtaaactga acatctacta ccgtcgtctg 3720

tacaacggtc tgaaattcat catcaaacgt tacaccccga acaacgaaat cgactgcttc 3780

gttaaatctg gtgacttcat caaactgtac gtttcttaca acaacaacga acacatcgtt 3840

ggttacccga aagacggtaa cgcgttcaac aacctggacc gtatcctgcg tgttggttac 3900

aacgcgccgg gtatcccgct gtacaaaaaa atggaagcgg ttaaactgcg tgacctgaaa 3960

acctactctg ttcagctgaa actgtacgac gacaaaaacg cgtctctggg tctggttggt 4020

acccacaacg gtcagatcgg taacgacccg aaccgtgaca tcctgatcgc gtctaactgg 4080

tacttcaacc acctgaaaga caaaatcctg ggttgcgact ggtacttcgt tccgaccgac 4140

gaaggttgga ccaacgacta aaagcttgat ccggctgcta acaaagcccg aaaggaagct 4200

gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg 4260

gtcttgaggg gttttttgct gaaaggagga actatatccg gatctggcgt aatagcgaag 4320

aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc 4380

cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 4440

ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 4500

ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 4560

tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 4620

cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 4680

tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 4740

ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 4800

attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg 4860

aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 4920

accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 4980

tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 5040

gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 5100

ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 5160

gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 5220

gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 5280

agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 5340

gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 5400

cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 5460

gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 5520

gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 5580

ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 5640

gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact 5700

ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 5760

tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 5820

actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 5880

taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 5940

gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 6000

tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 6060

ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 6120

gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc 6180

tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 6240

cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 6300

gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 6360

actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 6420

ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 6480

gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 6540

atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 6600

tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 6660

tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 6720

aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 6780

gcctctcccc gcgcgttggc cgattcatta atgcag 6816

<210> 16

<211> 1314

<212> PRT

<213> Escherichia coli

<400> 16

Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn Asp

1 5 10 15

Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile Tyr

20 25 30

Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu Arg

35 40 45

Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser Leu

50 55 60

Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr Asp

65 70 75 80

Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn Arg

85 90 95

Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile Asn

100 105 110

Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe Asp

115 120 125

Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro Ser

130 135 140

Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe Gly

145 150 155 160

Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu Arg

165 170 175

Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser Ile

180 185 190

Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn Val

195 200 205

Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe Gln

210 215 220

Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His Gly

225 230 235 240

Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys Gln

245 250 255

Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu Phe

260 265 270

Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys Asn

275 280 285

Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn Lys

290 295 300

Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp Ser

305 310 315 320

Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser Asn

325 330 335

Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn Ser

340 345 350

Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn Ile

355 360 365

Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys Ile

370 375 380

Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe Asn

385 390 395 400

Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met Arg

405 410 415

Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val Ser

420 425 430

Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile Arg

435 440 445

Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu

450 455 460

Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu Lys

465 470 475 480

Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn

485 490 495

Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile

500 505 510

Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr

515 520 525

Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser Asn

530 535 540

Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile Tyr

545 550 555 560

Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile Thr

565 570 575

Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr

580 585 590

Ser Tyr Phe Pro Ser Val Ile Cys Lys Val Asn Gln Gly Ala Gln Gly

595 600 605

Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn

610 615 620

Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser Thr

625 630 635 640

Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly Tyr

645 650 655

Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu Leu

660 665 670

Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu Ser

675 680 685

Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile Asp

690 695 700

Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys Leu

705 710 715 720

Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys Arg

725 730 735

Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys

740 745 750

Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu

755 760 765

Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu Lys

770 775 780

Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser Ser

785 790 795 800

Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln Leu

805 810 815

Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile Lys

820 825 830

Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser

835 840 845

Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser Lys

850 855 860

Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu

865 870 875 880

Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser

885 890 895

Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln

900 905 910

Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn Glu

915 920 925

Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn Asp

930 935 940

Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val

945 950 955 960

Ser Ala Cys His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile

965 970 975

Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val

980 985 990

Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly

995 1000 1005

Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn

1010 1015 1020

Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp

1025 1030 1035

Arg Leu Cys Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly

1040 1045 1050

Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn

1055 1060 1065

Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val

1070 1075 1080

Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys

1085 1090 1095

Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu

1100 1105 1110

Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr

1115 1120 1125

Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn

1130 1135 1140

Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Cys Tyr Thr Asn

1145 1150 1155

Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys

1160 1165 1170

Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Cys Phe

1175 1180 1185

Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn

1190 1195 1200

Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn

1205 1210 1215

Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile

1220 1225 1230

Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys

1235 1240 1245

Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser

1250 1255 1260

Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro

1265 1270 1275

Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu

1280 1285 1290

Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp

1295 1300 1305

Glu Gly Trp Thr Asn Asp

1310

<210> 17

<211> 6816

<212> DNA

<213> Escherichia coli

<400> 17

gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 60

agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat 120

catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac 180

gatgacgata aggatcgatg gggatccgag ctcgagccga tcaccatcaa caacttccgt 240

tactctgacc cggttaacaa cgacaccatc atcatgatgg aaccgccgta ctgcaaaggt 300

ctggacatct actacaaagc gttcaaaatc accgaccgta tctggatcgt tccggaacgt 360

tacgaattcg gtaccaaacc ggaagacttc aacccgccgt cttctctgat cgaaggtgcg 420

tctgaatact acgacccgaa ctacctgcgt accgactctg acaaagaccg tttcctgcag 480

accatggtta aactgttcaa ccgtatcaaa aacaacgttg cgggtgaagc gctgctggac 540

aaaatcatca acgcgatccc gtacctgggt aactcttact ctctgctgga caaattcgac 600

accaactcta actctgtttc tttcaacctg ctggaacagg acccgtctgg tgcgaccacc 660

aaatctgcga tgctgaccaa cctgatcatc ttcggtccgg gtccggttct gaacaaaaac 720

gaagttcgtg gtatcgttct gcgtgttgac aacaaaaact acttcccgtg ccgtgacggt 780

ttcggttcta tcatgcagat ggcgttctgc ccggaatacg ttccgacctt cgacaacgtt 840

atcgaaaaca tcacctctct gaccatcggt aaatctaaat acttccagga cccggcgctg 900

ctgctgatgc acgaactgat ccacgttctg cacggtctgt acggtatgca ggtttcttct 960

cacgaaatca tcccgtctaa acaggaaatc tacatgcagc acacctaccc gatctctgcg 1020

gaagaactgt tcaccttcgg tggtcaggac gcgaacctga tctctatcga catcaaaaac 1080

gacctgtacg aaaaaaccct gaacgactac aaagcgatcg cgaacaaact gtctcaggtt 1140

acctcttgca acgacccgaa catcgacatc gactcttaca aacagatcta ccagcagaaa 1200

taccagttcg acaaagactc taacggtcag tacatcgtta acgaagacaa attccagatc 1260

ctgtacaact ctatcatgta cggtttcacc gaaatcgaac tgggtaaaaa attcaacatc 1320

aaaacccgtc tgtcttactt ctctatgaac cacgacccgg ttaaaatccc gaacctgctg 1380

gacgacacca tctacaacga caccgaaggt ttcaacatcg aatctaaaga cctgaaatct 1440

gaatacaaag gtcagaacat gcgtgttaac accaacgcgt tccgtaacgt tgacggttct 1500

ggtctggttt ctaaactgat cggtctgtgc aaaaaaatca tcccgccgac caacatccgt 1560

gaaaacctgt acaaccgtac cgcgtctctg accgacctgg gtggtgaact gtgcatcaaa 1620

atcaaaaacg aagacctgac cttcatcgcg gaaaaaaact ctttctctga agaaccgttc 1680

caggacgaaa tcgtttctta caacaccaaa aacaaaccgc tgaacttcaa ctactctctg 1740

gacaaaatca tcgttgacta caacctgcag tctaaaatca ccctgccgaa cgaccgtacc 1800

accccggtta ccaaaggtat cccgtacgcg ccggaataca aatctaacgc ggcgtctacc 1860

atcgaaatcc acaacatcga cgacaacacc atctaccagt acctgtacgc gcagaaatct 1920

ccgaccaccc tgcagcgtat caccatgacc aactctgttg acgacgcgct gatcaactct 1980

accaaaatct actcttactt cccgtctgtt atctgcaaag ttaaccaggg tgcgcagggt 2040

atcctgttcc tgcagtgggt tcgtgacatc atcgacgact tcaccaacga atcttctcag 2100

aaaaccacca tcgacaaaat ctctgacgtt tctaccatcg ttccgtacat cggtccggcg 2160

ctgaacatcg ttaaacaggg ttacgaaggt aacttcatcg gtgcgctgga aaccaccggt 2220

gttgttctgc tgctggaata catcccggaa atcaccctgc cggttatcgc ggcgctgtct 2280

atcgcggaat cttctaccca gaaagaaaaa atcatcaaaa ccatcgacaa cttcctggaa 2340

aaacgttacg aaaaatggat cgaagtttac aaactggtta aagcgaaatg gctgggtacc 2400

gttaacaccc agttccagaa acgttcttac cagatgtacc gttctctgga ataccaggtt 2460

gacgcgatca aaaaaatcat cgactacgaa tacaaaatct actctggtcc ggacaaagaa 2520

cagatcgcgg acgaaatcaa caacctgaaa aacaaactgg aagaaaaagc gaacaaagcg 2580

atgatcaaca tcaacatctt catgcgtgaa tcttctcgtt ctttcctggt taaccagatg 2640

atcaacgaag cgaaaaaaca gctgctggaa ttcgacaccc agtctaaaaa catcctgatg 2700

cagtacatca aagcgaactc taaattcatc ggtatcaccg aactgaaaaa actggaatct 2760

aaaatcaaca aagttttctc taccccgatc ccgttctctt actctaaaaa cctggactgc 2820

tgggttgaca acgaagaaga catcgacgtt atcctgaaaa aatctaccat cctgaacctg 2880

gacatcaaca acgacatcat ctctgacatc tctggtttca actcttctgt tatcacctac 2940

ccggacgcgc agctggttcc gggtatcaac ggtaaagcga tccacctggt taacaacgaa 3000

tcttctgaag ttatcgttca caaagcgatg gacatcgaat acaacgacat gttcaacaac 3060

ttcaccgttt ctttctggct gcgtgttccg aaagtttctg cgtgccacct ggaacagtac 3120

ggtaccaacg aatactctat catctcttct atgaaaaaac actctctgtc tatcggttct 3180

ggttggtctg tttctctgaa aggtaacaac ctgatctgga ccctgaaaga ctctgcgggt 3240

gaagttcgtc agatcacctt ccgtgacctg ccggacaaat tcaacgcgta cctggcgaac 3300

aaatgggttt tcatcaccat caccaacgac cgtctgtgct ctgcgaacct gtacatcaac 3360

ggtgttctga tgggttctgc ggaaatcacc ggtctgggtg cgatccgtga agacaacaac 3420

atcaccctga aactggaccg ttgcaacaac aacaaccagt acgtttctat cgacaaattc 3480

cgtatcttct gcaaagcgct gaacccgaaa gaaatcgaaa aactgtacac ctcttacctg 3540

tctatcacct tcctgcgtga cttctggggt aacccgctgc gttacgacac cgaatactac 3600

ctgatcccgg ttgcgtcttc ttctaaagac gttcagctga aaaacatcac cgactacatg 3660

tacctgacca acgcgccgtg ctacaccaac ggtaaactga acatctacta ccgtcgtctg 3720

tacaacggtc tgaaattcat catcaaacgt tacaccccga acaacgaaat cgactgcttc 3780

gttaaatctg gtgacttcat caaactgtac gtttcttaca acaacaacga acacatcgtt 3840

ggttacccga aagacggtaa cgcgttcaac aacctggacc gtatcctgcg tgttggttac 3900

aacgcgccgg gtatcccgct gtacaaaaaa atggaagcgg ttaaactgcg tgacctgaaa 3960

acctactctg ttcagctgaa actgtacgac gacaaaaacg cgtctctggg tctggttggt 4020

acccacaacg gtcagatcgg taacgacccg aaccgtgaca tcctgatcgc gtctaactgg 4080

tacttcaacc acctgaaaga caaaatcctg ggttgcgact ggtacttcgt tccgaccgac 4140

gaaggttgga ccaacgacta aaagcttgat ccggctgcta acaaagcccg aaaggaagct 4200

gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg 4260

gtcttgaggg gttttttgct gaaaggagga actatatccg gatctggcgt aatagcgaag 4320

aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc 4380

cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 4440

ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 4500

ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 4560

tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 4620

cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 4680

tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 4740

ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 4800

attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg 4860

aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 4920

accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 4980

tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 5040

gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 5100

ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 5160

gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 5220

gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 5280

agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 5340

gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 5400

cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 5460

gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 5520

gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 5580

ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 5640

gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact 5700

ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 5760

tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 5820

actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 5880

taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 5940

gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 6000

tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 6060

ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 6120

gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc 6180

tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 6240

cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 6300

gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 6360

actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 6420

ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 6480

gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 6540

atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 6600

tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 6660

tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 6720

aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 6780

gcctctcccc gcgcgttggc cgattcatta atgcag 6816