阅读说明：本技术 光转换的多肽及其用途 (Photoconverted polypeptides and uses thereof ) 是由 A·斯克拉 A·赖歇特 M·道内 于 2018-05-09 设计创作，主要内容包括：本发明涉及光转换的多肽。具体而言,本发明涉及一种包含光响应元件的多肽,其中可以通过用特定波长的光照射该多肽而将该光响应元件的构型(即构型状态)在反式和顺式异构体之间转换,并且其中所述构型的转换改变了所述多肽的构象和与配体(例如目标分子)的结合活性。而且,本发明包括使用所述光转换多肽来分离和/或纯化目标分子。本发明还提供了包含本发明的光转换多肽的亲和基质、亲和色谱柱和亲和色谱设备。(The present invention relates to light-converting polypeptides. In particular, the present invention relates to a polypeptide comprising a light-responsive element, wherein the configuration (i.e. conformational state) of the light-responsive element can be switched between trans and cis isomers by irradiating the polypeptide with light of a specific wavelength, and wherein said switching of configuration changes the conformation of said polypeptide and the binding activity to a ligand (e.g. a target molecule). Furthermore, the present invention includes the use of the photoconversion polypeptide for isolating and/or purifying a target molecule. The invention also provides affinity matrices, affinity chromatography columns and affinity chromatography devices comprising the light-converting polypeptides of the invention.)

1. A polypeptide comprising a light-responsive element,

wherein the configuration of the light responsive element can be switched by irradiating the polypeptide with light of a specific wavelength,

and wherein the change in configuration alters the binding activity of the polypeptide to the ligand.

2. Use of a polypeptide comprising a light-responsive element according to claim 1 for the isolation and/or purification of a target molecule.

3. The light-responsive element-containing polypeptide of claim 1 or the use of claim 2, wherein the light-responsive element-containing polypeptide is part of a solid phase.

4. A method for isolating and/or purifying a target molecule, the method comprising the steps of:

(i) contacting a liquid phase comprising a target molecule with the light-converting polypeptide comprising a light-responsive element according to claim 1 or 3,

wherein the polypeptide comprising a light-responsive element is part of a solid phase,

and wherein the light responsive element is in a first configuration such that the polypeptide has a high affinity for the target molecule; and

(ii) (ii) illuminating the polypeptide comprising the light-responsive element with a wavelength that changes the light-responsive element to the second configuration such that the polypeptide has a reduced affinity for the target molecule compared to the affinity of step (i), and eluting the target molecule.

5. The polypeptide comprising a light-responsive element of claim 1 or 3, the use of claim 2, or the method of claim 4, wherein the polypeptide comprising a light-responsive element is streptavidin or a variant or mutein thereof comprising a light-responsive element.

6. The light-responsive element-containing polypeptide of any one of claims 1, 3 and 5, the use of any one of claims 2,3 and 5, or the method of claim 4 or 5, wherein the light-responsive element-containing polypeptide comprises or consists of:

(i) 2, the amino acid sequence of SEQ ID NO;

(ii)4, the amino acid sequence of SEQ ID NO;

(iii) 6, the amino acid sequence of SEQ ID NO;

(iv) 86 of SEQ ID NO;

(v) 20, the amino acid sequence of SEQ ID NO; wherein the residue at position 12 of SEQ ID NO 20 is replaced by a light responsive element;

(vi) 61 in SEQ ID NO; wherein the residue at position 13 of SEQ ID NO 61 is replaced by a photoresponsive element;

or

(vii) (ii) an amino acid sequence having at least 80% identity to any one of the amino acid sequences of (i) to (vi),

wherein the polypeptide comprises a light-responsive element, wherein the configuration of the light-responsive element can be switched by irradiating the polypeptide with light of a specific wavelength, and wherein the switching of the configuration changes the binding activity of the polypeptide to a ligand.

7. The polypeptide comprising a light-responsive element of any one of claims 1, 3, 5 and 6, the use of any one of claims 2,3, 5 and 6, or the method of any one of claims 4-6, wherein conversion of one configuration of the light-responsive element to another configuration changes the conformation or shape of a ligand-binding pocket or site of the polypeptide.

8. A polypeptide comprising a light-responsive element as claimed in any one of claims 1, 3 and 5 to 7, a use as claimed in any one of claims 2,3 and 5 to 7, or a method as claimed in any one of claims 4 to 7, wherein the light-responsive element is in or near a ligand-binding pocket or site of the polypeptide.

9. A polypeptide comprising a light-responsive element as claimed in any one of claims 1, 3 and 5 to 8, a use as claimed in any one of claims 2,3 and 5 to 8, or a method as claimed in any one of claims 4 to 8, wherein the light-responsive element is involved in binding of a ligand to the polypeptide.

10. The polypeptide comprising a light-responsive element of any one of claims 1, 3 and 5-9, the use of any one of claims 2,3 and 5-9, or the method of any one of claims 4-9, wherein the light-responsive element is

(i) In SEQ ID NO: 2. at amino acid position 96 of any one of 4, 8 and 10;

(ii) in SEQ ID NO: position 132 of either of 6 and 12;

(iii) at position 12 of SEQ ID NO: 20;

(iv) at position 13 of either of SEQ ID NOs 61 and 86;

(v) in an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs 2,4, 8 or 10, in the amino acid sequence of SEQ ID NOs: 2. at a homologous amino acid position at amino acid position 96 of 4, 8 or 10;

(vi) in an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO 6 or 12, in the amino acid sequence of SEQ ID NO:6 or 12 at a homologous amino acid position at amino acid position 132;

(vii) in an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO 20, in a sequence that is identical to the amino acid sequence of SEQ ID NO:20 at a homologous amino acid position at amino acid position 12;

(viii) in an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs 61 and 86, in a sequence that differs from the amino acid sequence of SEQ ID NOs:61 at amino acid position 13.

11. The polypeptide comprising a light-responsive element of any one of claims 1, 3 and 5-10, the use of any one of claims 2,3 and 5-10, or the method of any one of claims 4-10, wherein a polypeptide comprising a first configuration of light-responsive elements has a higher affinity for a ligand than a polypeptide comprising a second configuration of light-responsive elements.

12. The polypeptide comprising a light-responsive element of any one of claims 1, 3 and 5-11, the use of any one of claims 2,3 and 5-11, or the method of any one of claims 4-11, wherein the polypeptide comprising a first configuration of light-responsive elements has a high affinity for a ligand and the polypeptide comprising a second configuration of light-responsive elements has a low affinity for the ligand.

13. The polypeptide comprising a light-responsive element of any one of claims 1, 3 and 5 to 12, the use of any one of claims 2,3 and 5 to 12, or the method of any one of claims 4 to 12, wherein the light-responsive element comprises an azo group.

14. The polypeptide comprising a light-responsive element of any one of claims 1, 3 and 5-13, the use of any one of claims 2,3 and 5-13, or the method of any one of claims 4-13, wherein the light-responsive element comprises a photo-converted amino acid side chain.

15. The polypeptide comprising a light-responsive element of any one of claims 1, 3, and 5-14, the use of any one of claims 2,3, and 5-14, or the method of any one of claims 4-14, wherein the light-responsive element comprises an unnatural amino acid, wherein two isomers of the unnatural amino acid are capable of being converted with light of a particular wavelength.

16. The polypeptide comprising a light-responsive element of any one of claims 1, 3 and 5-15, the use of any one of claims 2,3 and 5-15, or the method of any one of claims 4-15, wherein the light-responsive element comprises

(i)3' -carboxyphenylazophenylalanine or a derivative thereof; or

(ii)4' -carboxyphenylazophenylalanine or a derivative thereof.

17. The polypeptide comprising a light-responsive element of any one of claims 1, 3 and 5-16, the use of any one of claims 2,3 and 5-16, or the method of any one of claims 4-16, wherein the isomers are trans and cis isomers.

18. The polypeptide comprising a light-responsive element of any one of claims 1, 3, and 5-17, the use of any one of claims 2,3, and 5-17, or the method of any one of claims 4-17, wherein the polypeptide comprising 3 '-carboxyphenylazophenylalanine or the trans isomer of 4' -carboxyphenylazophenylalanine has an increased affinity for a ligand as compared to a polypeptide comprising the cis isomer of 3 '-carboxyphenylazophenylalanine or 4' -carboxyphenylazophenylalanine.

19. The polypeptide comprising a light-responsive element according to any one of claims 1, 3 and 5 to 18, the use according to any one of claims 2,3 and 5 to 18, or the method according to any one of claims 4 to 18, wherein at least 70% of the polypeptide comprises the trans-isomer of the light-responsive element under visible light having a wavelength of 405-470 nm.

20. The polypeptide comprising a light-responsive element of any one of claims 1, 3 and 5-19, the use of any one of claims 2,3 and 5-19, or the method of any one of claims 4-19, wherein at least 85% of the polypeptide comprises the cis-isomer of the light-responsive element under Ultraviolet (UV) light having a wavelength of 310-370 nm.

21. A polypeptide comprising a light-responsive element as claimed in any one of claims 3 and 5 to 20, a use as claimed in any one of claims 3 and 5 to 20, or a method as claimed in any one of claims 4 to 20, wherein the solid phase is hydrophilic.

22. The polypeptide comprising a light-responsive element of any one of claims 3 and 5-21, the use of any one of claims 3 and 5-21, or the method of any one of claims 4-21, wherein the solid phase is a matrix, a hydrogel, a bead, a magnetic bead, a chip, a glass surface, a plastic surface, a gold surface, a silver surface, or a plate.

23. The polypeptide comprising a light-responsive element of claim 22, the use of claim 22, or the method of claim 22, wherein the substrate, hydrogel, bead, chip, glass surface, plastic surface, or plate is light-transmissive.

24. A polypeptide comprising a light-responsive element according to claim 22 or 23, a use according to claim 22 or 23, or a method according to claim 22 or 23, wherein the matrix, hydrogel or bead is the solid phase of an affinity chromatography column.

25. The light-responsive element-containing polypeptide of any one of claims 22-24, the use of any one of claims 22-24, or the method of any one of claims 22-24, wherein the matrix is N-hydroxysuccinimide (NHS) -activated CH-sepharose.

26. The polypeptide comprising a light-responsive element of claim 22, the use of claim 22, or the method of claim 22, wherein the plate is a microtiter well plate.

27. The polypeptide comprising a light-responsive element of any one of claims 1, 3 and 5-26, the use of any one of claims 2,3 and 5-26, or the method of any one of claims 4-26, wherein the polypeptide is covalently or non-covalently attached to a solid phase.

28. A polypeptide comprising a light-responsive element according to any one of claims 3 and 5 to 27, a use according to any one of claims 3 and 5 to 27, or a method according to any one of claims 4 to 27, wherein the solid phase is light-fast at least in the wavelength range of 300nm to 500nm, preferably 330nm to 450 nm.

29. The polypeptide comprising a light-responsive element of any one of claims 1, 3 and 5-28, the use of any one of claims 2,3 and 5-28, or the method of any one of claims 4-28, wherein the ligand is a molecule selected from the group consisting of: peptides, oligopeptides, polypeptides, proteins, antibodies or fragments thereof, immunoglobulins or fragments thereof, enzymes, hormones, cytokines, complexes, oligonucleotides, polynucleotides, nucleic acids, carbohydrates, liposomes, nanoparticles, cells, biological macromolecules, biomolecules, and small molecules.

30. The polypeptide comprising a light-responsive element of claim 29, the use of claim 29, or the method of claim 29, wherein the peptide ligand comprises or consists of:

(i) 13, the amino acid sequence of SEQ ID NO;

(ii) 14, the amino acid sequence of SEQ ID NO; or

(iii) An amino acid sequence having at least 80% identity to SEQ ID NO 13 or 14 and having affinity to streptavidin or a mutant or variant thereof.

31. The polypeptide comprising a light-responsive element of claim 30, the use of claim 30, or the method of claim 30, wherein the streptavidin mutant is a polypeptide having the amino acid sequence of SEQ ID NO:7, or a tetramer of proteins.

32. The method of any one of claims 4 to 31, wherein the polypeptide is irradiated with 400-500nm visible light before and/or during step (i).

33. The method of any one of claims 4-32, wherein the method further comprises the steps of:

(i') washing the solid phase with an appropriate buffer.

34. The method of claim 33, wherein the polypeptide is irradiated during step (i') with visible light having a wavelength of 400-500 nm.

35. The method of any one of claims 4-34, wherein the polypeptide is irradiated during step (ii) with ultraviolet light having a wavelength of 300-390 nm.

36. The method of any one of claims 4-35, wherein the method further comprises the steps of:

(iii) the polypeptide comprising the light-responsive element is regenerated into a first conformation having affinity for the target molecule.

37. The method of claim 36, wherein the photoresponsive element is regenerated during step (iii) by irradiating the polypeptide with 400-500nm visible light.

38. The method of claim 36 or 37, wherein the solid phase is washed with a suitable buffer during step (iii).

39. The use of any one of claims 2,3 and 5-31, or the method of any one of claims 4-38, wherein the target molecule is a molecule selected from the group consisting of: peptides, oligopeptides, polypeptides, proteins, antibodies or fragments thereof, immunoglobulins or fragments thereof, enzymes, hormones, cytokines, complexes, oligonucleotides, polynucleotides, nucleic acids, carbohydrates, liposomes, nanoparticles, cells, biological macromolecules, biomolecules, and small molecules.

40. The use of any one of claims 2,3, 5-31 and 39, or the method of any one of claims 4-39, wherein the target molecule is a native protein or a recombinantly produced protein.

41. The use of any one of claims 2,3, 5-31, 39 and 40, or the method of any one of claims 4-40, wherein the target molecule is a therapeutic protein.

42. The use of claim 39, or the method of claim 39, wherein the antibody fragment is a Fab fragment, a F (ab')2 fragment, a Fd fragment, a Fv fragment, a scFv fragment, or a single domain antibody.

43. The method of any one of claims 4-42, wherein the liquid phase comprising the target molecule is a cell extract or a culture supernatant.

44. The method of claim 43, wherein the cellular extract is a cytosolic or whole-cell extract.

45. An affinity matrix comprising a polypeptide comprising a light-responsive element according to any one of claims 1, 3 and 5-31.

46. An affinity chromatography column comprising the affinity matrix of claim 45.

47. The affinity chromatography column of claim 46, wherein the matrix is contained in a light-transmissive tube or container; and/or in a tube or container containing at least one optical fiber.

48. The affinity chromatography column of claim 47, wherein the light-transmissive tube or vessel is made of glass or plastic.

49. An affinity chromatography apparatus comprising

(i) An affinity chromatography column of any one of claims 46-48;

(ii) a light source;

(iii) a housing; and

(iv) an electronic interface.

50. The affinity chromatography apparatus of claim 49, wherein the light source comprises or consists of one, two or more light emitting diodes, LEDs, fluorescent tubes, and/or lasers.

51. An affinity chromatography apparatus as claimed in claim 49 or 50, wherein the wavelength of light emitted by the light source is electronically controlled.

52. The affinity chromatography apparatus of any one of claims 49-51, wherein the wavelength of light emitted by the light source is switchable.

53. The affinity chromatography device of any one of claims 49-52, wherein the wavelength of light emitted by the one, two or more light sources is convertible from visible light having 400-500nm to ultraviolet light having 300-390nm, and vice versa.

Description of the drawings:

FIG. 1: the principle of light-operated affinity chromatography for purifying protein.

The affinity column comprises a chromatography matrix with immobilized light-converting binding proteins (affinity molecules). A protein solution (e.g., a cell extract) is applied to the column and once the target protein (e.g., with an affinity tag such as Strep-tag II) is bound to the affinity matrix, contaminating proteins and biomolecules (possibly including host cells and/or any type of buffer components) are washed away. By irradiation with mild uv light at 365nm, the conformation of the binding protein in the affinity matrix will change, losing the binding activity to the target protein and/or affinity tag, thus allowing for immediate elution (under constant buffer flow). For subsequent regeneration of the chromatography column, green light >530nm may be applied, thereby relaxing the affinity matrix to the ground state.

FIG. 2: synthesis of the azo-benzene based photoconverted unnatural amino acid 4' -carboxyphenylazophenylalanine, alias 4- [ (4-carboxyphenyl) azo ] -L-phenylalanine.

(A) Preparation of 4- [ (4-carboxyphenyl) azo via Boc-or Fmoc-protected intermediate]L-phenylalanine (Caf; 7), also indicating reversible isomerization from the trans to the cis configuration, triggered by light of different wavelengths. (B)4- [ (4-carboxyphenyl) azo]-L-phenylalanine (7)At D₂In O¹H-NMR spectrum. (C)4- [ (4-carboxyphenyl) azo]L-phenylalanine (7) at D₂In O¹³C-NMR spectrum.

FIG. 3: synthesis of azo-benzene based photoconverted unnatural amino acid 3' -carboxyphenylazophenylalanine, alias 4- [ (3-carboxyphenyl) azo ] -L-phenylalanine

(A) Preparation of 4- [ (3-carboxyphenyl) azo]L-phenylalanine (11), also illustrating reversible isomerization from the trans to the cis configuration, initiated by different wavelengths of light. (B)4- [ (3-carboxyphenyl) azo]L-phenylalanine (11) at D₂In O¹H-NMR spectrum. (C)4- [ (3-carboxyphenyl) azo]L-phenylalanine (11) at D₂In O¹³C-NMR spectrum.

FIG. 4: reversible light conversion (isomerization) of Caf caused by alternating cycles of light irradiation at 365nm (uv) and 530nm (green) LED.

(A) Ultraviolet spectrum of unnatural amino acid Caf in water (solid line: trans isomer; dotted line: cis isomer). (B) Reversible photoconversion between trans and cis configurations visible over 3 cycles of absorbance change at about 340nm (pi → pi transition). High absorption at 335nm indicates the trans configuration, while low absorption at 335nm indicates the cis configuration of Caf, see fig (a). HPLC chromatogram of (C-D)7, absorbance before irradiation at λ 286nm (C), absorbance after ultraviolet irradiation (D) and absorbance after green irradiation (E). The chromatogram in panel (C) shows substantially pure trans isomer; the chromatogram in panel (D) shows that the cis isomer is predominant and the trans isomer is a minor species. The chromatogram in panel (E) shows that the trans isomer is predominant, while the cis isomer is a minor species.

FIG. 5: SAm1^CafStructural and sequence summaries of the variants.

(A) Crystal structure of the complex between streptavidin mutant 1(SAm1, Strep-Tactin) and Strep-tag II with highlighted residues V44, W108 and W120(PDB entry 1KL 3). The potential of all positions substituted with Caf to interfere with Strep-tag II binding (decrease affinity) in the cis configuration of the unnatural amino acid Caf, but to retain binding in its (trans) ground state was investigated. Among these positions studied for introducing Caf as a light responsive element, V44 and W120 are less preferred. (B) Nucleic acid and amino acid sequence of SAm1, highlighting the position of Caf introduction (in translation/suppression of the amber stop codon).

FIG. 6: SAm1^Caf108Expression, purification and refolding of

(A) plasmid map of pSBX8.CafRS #30d53(SEQ ID NO: 55). (B) Purification and refolding of recombinant core streptavidin mutant SAm1 carrying Caf at position 108. During the preparation of the recombinant protein, samples from different stages showed SDS-PAGE (15%) gels stained with Coomassie Brilliant blue. Lanes: 1, inducing total escherichia coli (e.coli) protein before gene expression; 2, total cellular protein 12h after induction; 3, inclusion body renaturation and protein solution after CEX purification; 4, same sample as 3, but without heat treatment prior to SDS-PAGE. Under these conditions, the core streptavidin tetramer remains intact (Bayer et al, 1990Methods enzymol.184: 80-89). Thus, the correct folding state of the recombinant mutant streptavidin in the final preparation was confirmed, while a small amount of monomeric (possibly non-functional) streptavidin was still present after refolding (lane 4). Lane 5 shows the same sample as lane 4, but at a lower concentration.

FIG. 7: reversible binding of PhoA/Strep-tag II fusion proteins to streptavidin mutants/variants (modified with light-converted amino acids) in ELISA.

(A) ELISA setup for screening streptavidin mutants with reversible binding activity to Strep-tag II peptide in response to UV light. (B) Purified PhoA/Strep-tag II was screened for light-induced desorption from SAm1 and its variants Caf44, Caf108, Caf 120. All streptavidin mutants tested showed good affinity for the PhoA/Strep-tag II fusion protein, giving rise to a visible light illumination signal comparable to SAm 1. In contrast, the streptavidin variant SAm1^Caf 108A significant decrease in the residual enzyme activity was observed after 365nm UV irradiation. This indicates that the streptavidin variant SAm1 was generated after light-induced conversion of Caf to cis configuration^Caf108The affinity to PhoA with Strep-tag II was reduced.

FIG. 8: photo-induced desorption of PhoA/Strep-tag II from the functionalized affinity matrix.

(A) In a sample containing 20. mu.L agarose and immobilized SAm1^Caf108The flow profile observed on the column of (1). Irradiation with green LED light (530nm) or mild ultraviolet light (365nm) as indicated. (B) Analysis by SDS-PAGE from SAm1^Caf108Samples of each fraction (10. mu.L) collected on the column. Lanes: m, molecular weight standard; l, loaded sample; FT, flow-through; w, washing; E1-E3, elution fraction. (C) 15% SDS-PAGE of samples from a SAm1 column. (D) The PhoA/Strep-tag II fusion proteins (sample loading, flow-through, wash, elution 1-3) in the pooled fractions were quantified by PhoA enzyme assay. In contrast to the unmodified streptavidin mutein (SAm1), it comprises SAm1^Caf108Shows light-dependent elution of the bound PhoA/Strep-tag II fusion protein.

FIG. 9: ProtL^CafStructural and sequence summaries of the variants.

(A) The crystal structure of the complex between the trastuzumab Fab fragment and the B1 domain of protein L, with Caf337 and mutated residues Asn361 and Ser365, is shown as a rod (UniProt accession No. Q51918; this corresponds to positions 29, 53 and 57 in PDB entry 4 HKZ). Position 337 is suitable for substitution with Caf in order to achieve different affinities for immunoglobulins depending on the cis or trans configuration of the light-responsive unnatural amino acid. (B) ProtL^CafNucleic acid and amino acid sequences of ABD fusion proteins highlighting position 337 for Caf introduction (translation/suppression with amber stop codon). And SEQ ID NO: in contrast to 20, methionine (underlined) was added as the start codon.

FIG. 10: ProtL^Caf337Expression and purification of the ABD fusion protein

SDS-PAGE (15%) gels stained with Coomassie Brilliant blue showed samples at various stages during recombinant protein preparation. Lanes: 1, inducing total escherichia coli (e.coli) protein before gene expression; 2, total cellular protein 12h after induction; 3, the insoluble fraction of the whole cell extract; 4, soluble supernatant of whole cell extract; 5,fractions eluted from HSA affinity chromatography; FIG. 6 ProtL after CEX purification^Caf337-ABD。

FIG. 11: immunoglobulins and ProtL in ELISA^CafReversible binding of ABD fusion proteins (modified with light-switched amino acids).

(A) For screening ProtL^CafSchematic ELISA setup of variants with reversible binding activity to immunoglobulins in response to uv light. (B) Exemplary assay of mouse anti-6 xHis antibodies (immunoglobulins) coupled with Alkaline Phosphatase (AP) by light-induced desorption from protein L domain B1 and its variant Caf337, both fused to ABD and adsorbed onto HSA coated microtiter plates. In its ground state, the Caf337 variant tested, although showing a lower signal compared to the unmodified protein L domain (left, open circles), still shows a high affinity for the IgG (right, open circles). In contrast, only ProtL was present after irradiation with 365nm UV light^Caf337The variant observed a significant decrease in the residual activity of the bound Ig-AP conjugate (filled circles), indicating that light-induced formation of the Caf cis isomer leads to specific dissociation between the light-switched protein L domain and the immunoglobulin (Ig).

Error bars represent the standard deviation of triplicate measurements. For ProtL in its ground state^Caf337Curve fitting of ELISA data (Voss)&Skerra1997Protein Eng 10:975-82) showed a dissociation constant of approximately 140nM for the complex formed with the anti-6 XHis antibody, corresponding to high affinity. The signal intensity observed after uv irradiation was too low to predict the dissociation constant, indicating that the affinity of the photoconversion polypeptide was greatly reduced (therefore, these data were fitted as a straight line).

The examples illustrate the invention.