阅读说明：本技术 新型细菌lpp突变体及其在重组蛋白质的分泌生产中的用途 (Novel bacterial lpp mutants and their use in the secretory production of recombinant proteins ) 是由 T·达斯勒 M·库尧 于 2018-07-24 设计创作，主要内容包括：本发明涉及一种细菌菌株,其含有至少一种编码重组蛋白质的基因,其特征在于其含有由以下组成的开放阅读框：(i)编码介导所述蛋白质向周质的易位的N-末端信号肽的DNA片段,其连接至(ii)编码脂蛋白(Lpp(N))的后续DNA序列(lpp(N)),其与野生型lpp基因编码的脂蛋白Lpp相比,具有至多十个氨基酸的差异,以及(iii)编码脂蛋白(Lpp(C))的另一DNA序列(lpp(C)),其与野生型lpp基因编码的脂蛋白(Lpp)相比,具有至多十个氨基酸的差异。本发明还涉及一种用于使用根据本发明的细菌菌株发酵生产重组蛋白质的方法。如此,可能实现与现有技术相比培养基中的重组蛋白的增加的量。(The invention relates to a bacterial strain containing at least one gene coding for a recombinant protein, characterized in that it contains open reading frames consisting of: (i) a DNA fragment encoding an N-terminal signal peptide mediating translocation of the protein to the periplasm linked to (ii) a subsequent DNA sequence (Lpp (N)) encoding a lipoprotein (Lpp (N)) which differs by at most ten amino acids compared to the lipoprotein Lpp encoded by the wild-type Lpp gene, and (iii) another DNA sequence (Lpp (c)) encoding a lipoprotein (Lpp (c)) which differs by at most ten amino acids compared to the lipoprotein (Lpp) encoded by the wild-type Lpp gene. The invention also relates to a method for the fermentative production of recombinant proteins using the bacterial strain according to the invention. In this way, it is possible to achieve an increased amount of recombinant protein in the culture medium compared to the prior art.)

1. A bacterial strain comprising at least one gene encoding a recombinant protein, wherein said bacterial strain comprises open reading frames consisting of:

i a DNA fragment encoding an N-terminal signal peptide mediating translocation of the protein to the periplasm, linked to

ii a subsequent DNA sequence (Lpp (N)) encoding a lipoprotein (Lpp (N)) which differs by at most ten amino acids compared to the lipoprotein Lpp encoded by the wild-type Lpp gene, and

iii another DNA sequence (Lpp (C)) encoding a lipoprotein (Lpp (C)) which differs by at most ten amino acids compared to the lipoprotein (Lpp) encoded by the wild-type Lpp gene.

2. The bacterial strain of claim 1, wherein the bacterial strain is a strain of the () species of escherichia coli.

3. A bacterial strain according to any one or both of claims 1 or 2, which does not contain other genes encoding proteins having at least 80% identity compared to the processed wild-type Lpp protein.

4. Bacterial strain according to one or more of claims 1 to 3, characterized in that Lpp (N) and Lpp (C) are connected by a linker consisting of one to more than one amino acid.

5. Bacterial strain according to one or more of claims 1 to 4, characterized in that the amino acid sequences encoded by Lpp (N) and Lpp (C) differ from the amino acid sequence of the wild-type Lpp protein by:

for Lpp (N), the C-terminal amino acid lysine present in wild-type Lpp protein is absent, or

For Lpp (C), the N-terminal amino acid cysteine present in wild-type Lpp protein is absent.

6. Bacterial strain according to one or more of claims 1 to 5, characterized in that in at least one of the amino acid sequences encoded by Lpp (N) or Lpp (C) in place of arginine at amino acid position 77 any other proteinogenic amino acid is present (based on the numbering and sequence of the amino acids of the unprocessed wild-type Lpp protein).

7. The bacterial strain of claim 6, wherein the proteinogenic amino acid at position 77 in at least one of the amino acid sequences encoded by lpp (N) or lpp (C) is cysteine.

8. Bacterial strain according to one or more of claims 1 to 7, characterized in that the N-terminal signal peptide is an amino acid sequence with at least 80% identity with respect to the signal peptide of the wild-type Lpp protein.

9. Bacterial strain according to claim 8, characterized in that the N-terminal signal peptide contains some other proteinogenic amino acid than glycine at amino acid position 14 and is identical to the signal peptide of the wild-type Lpp protein at all other amino acid positions (based on the amino acid numbering and sequence of the signal peptide of the wild-type Lpp protein).

10. The bacterial strain of claim 9, wherein the proteinogenic amino acid at position 14 of the N-terminal signal peptide is aspartic acid.

11. Bacterial strain according to one or more of claims 1 to 10, characterized in that the amino acids forming the linker sequence are selected from the group consisting of: glycine, serine and alanine.

12. Bacterial strain according to one or more of claims 1 to 11, characterized in that the open reading frame encoding the 2xLpp protein is located in the chromosome and the open reading frame encoding the wild-type Lpp protein is not located in the chromosome.

13. Bacterial strain according to one or more of claims 1 to 12, characterized in that the recombinant protein is a heterologous protein.

14. A process for the fermentative production of recombinant proteins, characterized in that a bacterial strain according to one or more of claims 1 to 13 is cultivated in a fermentation medium, the fermentation medium is separated from the cells after fermentation, and the protein is isolated from the fermentation medium.

15. The method of claim 14, wherein the recombinant protein is purified from the fermentation medium after isolating the fermentation medium.

Examples

The following examples serve to further illustrate the invention without limiting it.

All molecular biological and microbiological methods used, such as Polymerase Chain Reaction (PCR), gene synthesis, isolation and purification of DNA, modification of DNA by restriction enzymes, Klenow fragments and ligases, transformation, P1 transduction and the like, are carried out in a manner known to the person skilled in the art, are described in the literature or recommended by the corresponding manufacturers. The oligonucleotides used were purchased from Metabion International AG (Planegg/Germany).

Example 1: escherichia coli JE5512 strain (2 xLpp. DELTA. mutant) producing an Lpp-forming fusion protein (2 xLpp. DELTA. protein)

1.Production of E.coli Strain JE5512lpp cat-sacB pKD46

The starting strain for the production of strains which form Lpp fusion proteins is the E.coli Lpp wild-type strain JE5512(HfrC man pps) (Hirota et al, 1977, Proc. Natl. Acad. Sci. USA 74, p. 1417-.

In this strain, the coding region of the chromosomal wild-type lpp gene (nucleotides 1 to 237 in SEQ ID No. 1) was first replaced by an expression cassette which contains not only the gene for chloramphenicol acetyltransferase (cat; UniProt No. P62577) but also the gene for levansucrase from Bacillus subtilis (SacB; UniProt No. P05655). For this purpose, used as template for the amplification of the cat-sacB cassette by means of PCR are derivatives of the plasmid pKO3 (Link et al, 1997, J.Bacteriol.179, pp 6228-6237; for the sequence of pKO3 see http:// arep.med.harvard.edu/labgc/pKO3v.html), wherein most of the region between the cat gene and the sacB gene has been removed by cleavage with the restriction enzymes SmaI and Bst1107I and religation of fragments of 4729 base pairs in size. The resulting plasmid was designated pKO 3-Delta-M13. PCR was performed to amplify the cat-sacB cassette using pKO3-Delta-M13 and the oligonucleotides lpp-cat-sac-fw (SEQ ID No.6) and lpp-cat-sac-rev (SEQ ID No.7) as templates. In this case, the first 60 nucleotides of lpp-cat-sac-fw are homologous to the sequence of the Open Reading Frame (ORF) of lpp on the 5 'side, and the first 60 nucleotides of lpp-cat-sac-rev are homologous to the sequence of the lpp ORF on the 3' side. The result is a linear DNA fragment containing the cat-sacB cassette.

The strain JE5512 was transformed with the plasmid pKD46(Coli Genetic Stock Center CGSC #:7739) and this gave the strain JE5512 pKD 46. Competent cells of strain JE5512 pKD46, which were generated according to the information of Datsenko and Wanner (2000, Proc. Natl. Acad. Sci. USA 97, page 6640-6645), were transformed with a linear DNA fragment containing the cat-sacB cassette. Integration of the cat-sacB cassette into the wild-type lpp ORF position of the JE5512 chromosome was selected on LB agar plates containing 20mg/L chloramphenicol. Obtained in this way are cells in which the wild-type lpp ORF has been completely replaced by the cat-sacB cassette (JE5512 lpp:: cat-sacB pKD 46). The integration achieved at the correct position in the chromosome was confirmed by PCR using the oligonucleotides pykF (SEQ ID No.8) and ynhG2(SEQ ID No.9) and chromosomal DNA from chloramphenicol resistant cells as templates. Then, cells of strain JE5512Lpp:: cat-sacB pKD46 express the gene cat encoding chloramphenicol acetyltransferase and the gene sacB encoding levansucrase instead of the Lpp wild-type gene.

2.Production of DNA fragments encoding 2 xLpp. DELTA.proteins

DNA fragments encoding the 2 xLpp. DELTA.protein were produced by the method of "overlap extension" PCR (Horton et al, 2013, BioTechniques 54,129-33). For this purpose, chromosomal DNA of JE5512 was first used as a template. This contains the wild-type lpp gene.

To produce a polypeptide containing inter alia lpp (N) Δ Lys₇₈The DNA fragment of (1) was subjected to PCR (PCR1) using the oligonucleotides lpp-Allel-fw (SEQ ID No.10) and lpp-2x-rev2(SEQ ID No. 11). A second PCR (PCR2) was performed using the oligonucleotides lpp-Allel-fw (SEQ ID No.10) and lpp-2x-rev3(SEQ ID No.12) using the product of PCR1 as a template, resulting in an extension of the PCR1 product of 41 base pairs on the 3' side.

To generate toIn particular comprising lpp (C) Δ Cys₂₁The DNA fragment of (1) was subjected to PCR (PCR3) using the oligonucleotides lpp-2x-fw2(SEQ ID No.13) and lpp-Allel-rev (SEQ ID No.14) and the chromosomal DNA of JE5512 as a template. An additional PCR (PCR4) was performed using the oligonucleotides lpp-2x-fw3(SEQ ID No.15) and lpp-Allel-rev (SEQ ID No.14) using the PCR3 product as a template, resulting in an extension of 57 base pairs on the 5' side of the PCR3 product.

Using the products of PCR2 and PCR4 as templates, a fifth PCR was finally performed using the oligonucleotides lpp-Allel-fw (SEQ ID No.10) and lpp-Allel-rev (SEQ ID No.14) as primers. The DNA fragment thus generated, which is 1005 base pairs in length, contains in particular the signal Sequence (SP) of the Lpp gene in the ORF, which is linked (in the present example) to the coding sequence of the Lpp wild-type protein containing the deletion of the lysine residue codon at position 78 (designated Lpp (N) Δ Lys-₇₈) The DNA fragment of (1), (in this example) a linker sequence (L) consisting of three consecutive glycine codons and (in this example) the coding sequence of the Lpp wild-type protein comprising a codon for the deletion of the cysteine residue at position 21 (designated Lpp (C) < delta > Cys >₂₁) And, in addition, about 300 base pairs of the 5 'and 3' regions, respectively, of the lpp locus (SEQ ID No.16, 2 xlpp. DELTA.). The 2xLpp delta protein formed by the DNA fragment is shown in figure 1C.

3. Production of E.coli Strain JE 55122 xlpp. DELTA.

In the next step, the cat-sacB cassette is replaced with 2 xlpp. DELTA. For this purpose, the product from PCR5 was transformed into competent cells of strain JE5512 lpp:cat-sacB pKD46 by the method of Datsenko and Wanner (see above). Integration of the start position of the wild-type lpp gene of chromosome that allows 2 xlpp. DELTA.into JE5512 lpp:cat-sacB pKD46 was selected on LB agar plates containing 7% sucrose. Since only cells that do not express sacB can be grown on sucrose-containing medium, cells can be selected in which the cat-sacB cassette has been replaced by 2 xlpp. DELTA. (JE 55122 xlpp. DELTA.). The integration at the correct position in the chromosome was confirmed by PCR using the oligonucleotides pykF (SEQ ID No.8) and ynhG2(SEQ ID No.9) and chromosomal DNA of sucrose-resistant cells as templates. The sequence of the integrated 2xlpp Δ was verified by sequencing of the PCR products. The resulting strain was named JE 55122 xlpp. DELTA.

Example 2: production of E.coli JE5512 Strain containing the lpp3 allele (lpp3 mutant)

For comparison purposes, an Lpp mutant containing the known Lpp3 allele rather than the chromosomal Lpp wild-type gene was generated from E.coli strain JE5512 (Giam et al, 1984, Eur. J. biochem.141, pp. 331-379). The Lpp3 allele is characterized by a mutation that results in an amino acid substitution of glycine to aspartic acid at position 14 of the Lpp protein and results in some "leakage" of the periplasmic protein by the cell (see US 2008/0254511 a 1).

PCR was performed on chromosomal DNA of the lpp3 mutant E.coli "W3110 lpp 3" using the oligonucleotides lpp-Allel-fw (SEQ ID No.10) and lpp-Allel-rev (SEQ ID No.14) (see US 2008/0254511A 1, example 3). The PCR product containing the lpp3 allele was integrated into the chromosome of strain JE5512 lpp:cat-sacB pKD46, similar to 2 xlpp. DELTA.as described in example 1. Correct integration of the PCR product into the chromosome was confirmed by PCR and sequencing as described above. The resulting strain was named JE5512lpp 3.

Example 3: fermentative production of Fab antibody fragments on a 3l Scale Using a 2 xlpp.DELTA.mutant

1. Production of plasmid pJF118ut-CD154

This example describes the generation of Fab fragments of the humanized monoclonal anti-CD 154 antibody 5c8 by means of the E.coli JE 55122 xlpp. delta. strain, the sequence of which is disclosed in Karpuss et al, (2001, Structure 9, pp. 321-. The plasmid pJF118ut described in US 2008/0254511 a1 was used as a starting vector for cloning and expressing genes for anti-CD 154 Fab fragments. pJF118ut was deposited with the DSMZ-German centre for microorganism and Cell culture (German Collection of Microorganisms and Cell Cultures GmbH) (Braunschweig) under accession number DSM 18596. The two reading frames (including the signal sequence in each case) of the heavy chain (VH-CH1 domain) and the light chain (VL-CL domain) of the Fab fragment were cloned into the plasmid. For this purpose, the following steps are carried out: a DNA fragment having SEQ ID No.17 was generated by means of gene synthesis (Eurofins Genomics). Which comprises a gene fusion consisting of:

i a signal sequence derived from SEQ ID No.5, and

ii the reading frame of the heavy chain of the Fab fragment,

and a gene fusion consisting of:

i PhoA signal sequence of Escherichia coli and

ii reading frame of the light chain of a Fab fragment and

iii a linker consisting of four amino acids at the C-terminus of the light chain and

iv a hexa-histidine tag at the C-terminus of the linker.

The DNA fragment was cleaved with restriction enzymes EcoRI and PdmI and ligated with the expression vector pJF118ut which had been cleaved with EcoRI and SmaI. The resulting plasmid was designated pJF118ut-CD154, where the expression of the heavy and light chain genes of the anti-CD 154 Fab fragment was controlled by the tac promoter. FIG. 2 shows a plasmid map of plasmid pJF118ut-CD 154.

2.Production of anti-CD 154 Fab antibody fragments

For the production of anti-CD 154 Fab antibody fragments on a fermenter scale, CaCl was used₂Methods strains JE5512, JE5512lpp3 and JE 55122 xlpp. DELTA.were transformed with plasmid pJF118ut-CD 154. Selection of plasmid-containing cells was carried out with the aid of tetracycline (20 mg/l).

Production was carried out in a 3l stirred tank fermenter.

1.2l of an inorganic salt medium commonly used for culturing E.coli, comprising 15g/l glucose and enriched with complex components (1.5g/l Hy-Express II (Kerry); 1.0g/l Amisoy (Kerry); 0.5g/l Hy-Yest (Kerry))) the preliminary culture (which had been shake-cultured in complex medium (30g/l phytopeptones (BD Biosciences), 5g/l yeast extract (Oxoid), 5g/l NaCl) at 30 ℃ for about 6h) to about OD₆₀₀0.01. Inoculation represents time point 0 of fermentation or start of fermentation. During the fermentation, the temperature was set at 30 ℃ and NH was metered in₄OH or H₃PO₄The pH was kept constant at 7.0. Initially, cultures were stirred at 400rpm and sterilized through sterile filters at 2vvmThe compressed air of the bacteria is bubbled. Under these starting conditions, the oxygen probe was calibrated to 100% saturation already prior to seeding. The O in the fermentation process₂The target value of saturation was set to 30%. Once O is present₂The saturation drops below the target value and the regulation cascade is started to bring O₂The saturation returns to the target value. In this respect, the gas supply was first continuously increased to a maximum of 5vvm and then the stirring speed was continuously increased to a maximum of 1500 rpm. Glucose addition was started 10 hours after the start of the culture. The temperature was reduced from 30 ℃ to 27 ℃ about 0.5-1h before the planned induction. After a cultivation time of about 21-23h, isopropyl beta-D-thiogalactopyranoside (IPTG) was added to 0.1mM to achieve inducible expression.

After an incubation time of 64h, samples were collected, cells were removed from the medium by centrifugation and the content of Fab fragments in the culture supernatant was determined by means of a sandwich ELISA assay (see below). To determine the amount of target protein in the whole culture broth, i.e. the sum of the intracellular and extracellular Fab fragments present, the Fab content in the homogenized culture broth was determined. For this, 150. mu.l of the culture broth was mixed with 850. mu.l of 100mM Tris/Cl buffer (pH 7.4) and the cells were disrupted using a FastPrep homogenizer (FastPrep-24TM 5G, MP Biomedicals). After removal of cell debris by centrifugation, the clarified supernatant was used for a sandwich ELISA assay.

The anti-CD 154 Fab fragments were quantified by a sandwich ELISA assay known to those skilled in the art. This involves The use of immobilized anti-Fd heavy chain antibody (The Binding Site, product number: PC075) as capture agent and an oxidase-conjugated goat anti-human kappa light chain antibody (Sigma, product number: A7164) as detection antibody. Quantification is achieved by converting the chromogenic substrate Dako TMB + (Dako, product number: S1599) by peroxidase and the associated change in absorbance at 450 nm. The ELISA was calibrated using the Fab fragment "Human Fab/Kappa" (Bethy Laboratories, product No.: P80-115).

Table 1 lists the production of anti-CD 154 antibody fragments in the culture supernatants and in the whole culture.

Table 1: anti-CD 154 titres in culture supernatants and whole cultures after 64h fermentation

Example 4: fermentative production of Cyclodextrin glycosyltransferase Using 2 xlpp.DELTA.mutant on 3l Scale

For the production of cyclodextrin glycosyltransferase (CGTase) on a 3l scale, CaCl was used₂Methods strains JE5512, JE5512lpp3 and JE 55122 xlpp Δ were transformed with plasmid pCGT. Selection of plasmid-containing cells was performed with the aid of tetracycline (20 mg/l).

The production of plasmid pCGT for CGTase overexpression is described in example 4 of US 2008/0254511 a1 and the plasmid map is shown in figure 4 of US 2008/0254511 a 1. In essence, the plasmid contains not only the tetracycline resistance gene but also a structural gene (including a native CGTase signal sequence) of CGTase, particularly from Klebsiella pneumoniae. The expression of the gene encoding CGTase is regulated by the tac promoter.

The cultures for fermentative production of CGTase were carried out as described in example 3 using the strains JE5512/pCGT, JE5512lpp 3/pCGT and JE 55122 xlpp. DELTA./pCGT.

After 64 hours of fermentation, samples were collected and the CGTase content in the culture supernatant and in the homogenized and clarified broth was then determined by means of the CGTase activity assay based on the amount of Cyclodextrin (CD) enzymatically produced from starch (see example 3).

Determination of CGTase Activity

Determination of buffer: 5mM Tris HCl buffer, 5mM CaCl₂ x 2H₂O，pH 6.5

Substrate solution: 10% starch solution in assay buffer (Merck No.1.01252), pH 6.5

The mixture was measured: 0.2ml substrate solution +0.2ml appropriately diluted CGTase sample (culture supernatant or homogenized and clarified broth)

Reaction temperature: 40 deg.C

Enzyme assay:

pre-conditioning the temperature of the substrate solution and the CGTase-containing sample (about 5min at 40 ℃ C.)

Preparing an assay mixture by rapidly mixing (vortex mixer) the substrate solution and the CGTase-containing sample, if necessary diluting the sample with assay buffer in order to determine the value of 0.9-1.5g/l CD in the subsequent HPLC analysis;

incubate at 40 ℃ for 3 min

The enzymatic reaction was stopped by adding 0.6ml of methanol and mixing rapidly (vortex mixer)

Cool the mixture on ice (about 5min)

Centrifuge (5min, 12000rpm) and aspirate clear supernatant

Analysis of the amount of CD produced by HPLC: the analysis was carried out on an Agilent HP 1100HPLC system with a Nucleodur 100-3NH2-RP column (150 mm. times.4.6 mm, Macherey-Nagel) and 64% aqueous acetonitrile (v/v) as mobile phase, at a flow rate of 2.1 ml/min. Detection was performed by RI detector (1260Infinity RI, Agilent) and quantitation was performed based on peak area and alpha-CD standard (Cavamax W6-8 Pharma, Wacker).

Calculating enzyme activity: a ═ G × V1 ═ V2/(t × MG) [ U/ml ]

A is the activity of the compound (I),

content of G ═ CD (mg/l)

V1-determination of dilution factor in mixture

V2-dilution factor of the CGT-containing sample prior to use in the assay; if undiluted, then: v2 ═ 1

time of reaction (min)

MG ═ molecular weight (g/mol) (MG_CD＝973g/mol)

1 Unit (U)Product (CD)/min

Table 2 shows the respective CGTase yields obtained.

Table 2: CGTase yield in culture supernatant after 64h fermentation

The proportion of CGTase released into the medium by JE 55122 xlpp. DELTA./pCGT compared to total protein was similar to that of anti-CD 154 Fab-about 58%.

Examples 3 and 4 describe the fermentative production of medically relevant Fab antibody fragments and industrial enzymes, respectively, using various strains of e. In both cases, the 2xlpp Δ mutant according to the invention shows superiority with respect to the amount of target protein released into the culture medium compared to the Lpp wild-type strain and to the Lpp3 mutant with the "leaky phenotype".

Sequence listing

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<400> 16

atttgtatat cgaagcgccc tgatgggcgc tttttttatt taatcgataa ccagaagcaa 60

taaaaaatca aatcggattt cactatataa tctcacttta tctaagatga atccgatgga 120

agcatcctgt tttctctcaa tttttttatc taaaacccag cgttcgatgc ttctttgagc 180

gaacgatcaa aaataagtgc cttcccatca aaaaaatatt ctcaacataa aaaactttgt 240

gtaatacttg taacgctaca tggagattaa ctcaatctag agggtattaa taatgaaagc 300

tactaaactg gtactgggcg cggtaatcct gggttctact ctgctggcag gttgctccag 360

caacgctaaa atcgatcagc tgtcttctga cgttcagact ctgaacgcta aagttgacca 420

gctgagcaac gacgtgaacg caatgcgttc cgacgttcag gctgctaaag atgacgcagc 480

tcgtgctaac cagcgtctgg acaacatggc tactaaatac cgcggtggcg gaagctctaa 540

cgccaaaatt gatcaactga gcagcgatgt gcagaccctg aatgcgaaag tggatcagct 600

gtcaaacgat gttaatgcaa tgcgtagcga tgtgcaggca gcaaaggatg atgcagcacg 660

cgcaaatcag cgcctggata atatggcaac caagtatcgt aaataatagt acctgtgaag 720

tgaaaaatgg cgcacattgt gcgccatttt ttttgtctgc cgtttaccgc tactgcgtca 780

cgcgtaacat attcccttgc tctggttcac cattctgcgc tgactctact gaaggcgcat 840

tgctggctgc gggagttgct ccactgctca ccgaaaccgg ataccctgcc cgacgataca 900

acgctttatc gactaacttc tgatctacag ccttattgtc tttaaattgc gtaaagcctg 960

ctggcagtgt gtatggcatt gtctgaacgt tctgctgttc ttctg 1005

<210> 17

<211> 1599

<212> DNA

<213> artificial

<220>

<221> misc_feature

<222> (25)..(1549)

<223> anti-CD154 expression cassette

<220>

<221> sig_peptide

<222> (25)..(114)

<223> AFA signal sequence

<220>

<221> misc_feature

<222> (115)..(777)

<223> reading frame for HC (VH-CH1)

<220>

<221> sig_peptide

<222> (800)..(862)

<223> phoA signal sequence

<220>

<221> misc_feature

<222> (863)..(1516)

<223> reading frame for LC (VL-CL)

<220>

<221> misc_feature

<222> (1517)..(1546)

<223> linker including His tag

<400> 17

acagaattct aaggaggaaa ttatatgaaa agaaaccgtt tttttaatac ctcggctgct 60

attgccattt cgattgcatt acagatcttt tttccgtccg cttccgcttt cgctcaggtt 120

cagctggtgc agagcggtgc cgaagttgtt aaaccgggtg caagcgttaa actgagctgt 180

aaagcaagcg gctatatttt taccagctat tatatgtatt gggtgaaaca ggcaccgggt 240

cagggtctgg aatggattgg tgaaattaat ccgagcaatg gcgataccaa ttttaatgaa 300

aaatttaaaa gcaaagccac cctgaccgtt gataaaagcg caagcaccgc atatatggaa 360

ctgagcagcc tgcgtagcga agataccgca gtttattatt gtacccgtag tgatggtcgc 420

aatgatatgg atagctgggg tcagggcacc ctggtcaccg ttagcagcgc aagcaccaaa 480

ggtccgagcg tttttccgct ggcaccgagc agcaaaagca ccagcggtgg caccgcagca 540

ctgggttgtc tggttaaaga ttattttccg gaaccggtta cagttagctg gaatagcggt 600

gcactgacca gtggtgttca tacctttccg gcagttctgc aaagcagcgg tctgtatagc 660

ctgagcagcg ttgttaccgt tccgagcagt agcctgggca cccagaccta tatttgtaat 720

gttaatcata aaccgagcaa caccaaagtg gataaaaaag ttgaaccgaa aagctgctaa 780

taaccatgga gaaaataaag tgaaacaaag cactattgca ctggcactct taccgttact 840

cttcacccct gttaccaaag ccgatattgt gctcacccag agtccggcaa ccctgagcgt 900

tagtccgggt gaacgtgcaa ccattagctg tcgtgcaagc cagcgtgtta gcagcagcac 960

ctatagctat atgcattggt atcagcagaa accgggtcag cctccgaaac tgctgattaa 1020

atatgcaagc aatctggaaa gcggtgttcc ggcacgtttt agcggtagcg gtagtggcac 1080

cgattttacc ctgaccatta gcagcgttga accggaagat tttgccacct attattgtca 1140

gcatagctgg gaaattcctc cgacctttgg tggtggcacc aaactcgaga ttaaacgtac 1200

cgttgcagca ccgagcgtgt ttatctttcc gcctagtgat gaacagctga aaagcggcac 1260

cgcaagcgtt gtttgtctgc tgaataactt ttatccgcgt gaagcaaaag ttcagtggaa 1320

agttgataat gcactgcaaa gcggtaatag ccaagaaagc gttaccgaac aggatagcaa 1380

agatagcacc tatagcctgt caagcaccct gaccctgagc aaagcagatt atgaaaaaca 1440

caaagtgtat gcctgcgaag ttacccatca gggtctgagc agtccggtta caaaaagttt 1500

taatcgtggt gaatgtagct cttctgccca tcaccaccat caccattaat aagcttctag 1560

aagcttggct gttttggcgg atgagagaag attttcgac 1599