Chemically modified engineered spider silk proteins

Abstract

The present invention relates to recombinant spider silk protein bioconjugates (chemically modified engineered spider silk proteins). More specifically, the present invention relates to recombinant spider silk protein bioconjugates with polyethylene glycol polymers and their use in regenerative medicine.

Description

Technical Field

[0001] The present invention relates to recombinant spider silk protein bioconjugates (chemically modified engineered spider silk proteins). More specifically, the present invention relates to recombinant spider silk protein bioconjugates with polyethylene glycol polymers and their use in regenerative medicine. Background Art

[0002] Spider silk is made of proteins (spidroin), which usually consists of three structural subunits, namely non-repetitive N-terminal and C-terminal domains (NT and CT) and a long central region with highly repetitive sequences (REP). NT and CT are related to the formation process of silk, while the central region determines the mechanical properties of silk fibers.

[0003] Recombinant spidroin proteins can be produced using heterologous expression in bacteria, yeast, and other expression systems. Recombinant spidroin proteins generally differ from natural spidroin proteins in that they contain a smaller number of repeat sequences (Rep) in the central repeat region and generally lack one or both terminal domains.

[0004] The structural transformation of spider silk proteins from soluble liquid to solid fibers is mediated by changes in the pH and ion composition of the aqueous environment and shear forces. Studies on the terminal domain have shown that when the pH is reduced to about 5.5, NT dimerizes and becomes stable, thereby firmly connecting spider silk proteins to each other, while CT unfolds and forms amyloid fibrils, which can serve as nucleation seeds to promote the transformation of the repeat region into a β-sheet structure (Andersson et al., PLoS Biol. 2014, 12, e1001921).

[0005] Recombinant spider silk proteins lacking one or both terminal domains cannot reproduce the molecular mechanism of natural silk spinning, especially the structural transformation of the terminal domains. Therefore, converting them into β-sheet structures requires non-native methods, such as coacervation. Obtaining artificial spider silk in a biomimetic way requires recombinant spider silk proteins containing these two terminal domains and accurately reproducing the conditions in the spider silk gland to ensure the assembly of recombinant spider silk proteins.

[0006] EP 3263593 describes recombinant spider silk proteins containing these two terminal domains and capable of polymerizing in a biomimetic manner to form spider silk-like fibers, as well as methods for producing such fibers.

[0007] The present invention aims to circumvent the requirement for two terminal domains in the genetic structure of recombinant spider silk proteins, thereby still being able to generate spider silk-like fibers in a biomimetic manner. Summary of the invention

[0008] In a first aspect, the present invention relates to a recombinant spider silk protein comprising 244 to 620 amino acid residues and having a formula comprising three features: NT-(Rep) x -C defines:

[0009] aN-terminal domain NT, a fragment of 130 to 156 amino acid residues derived from the N-terminal domain of spider silk protein

[0010] composition;

[0011] b. Repeat domain (Rep) x , composed of 87 to 463 amino acid residues derived from repetitive sequences in spider silk proteins;

[0012] Where x is the number of repeating sequences; (Rep) x - the domain preferably consists of 174-463 amino acid residues;

[0013] c. Domain C, which is a Cys(Z) residue, wherein Z is optional and, if present, is selected from the group consisting of: Ala, Arg,

[0014] Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, The,

[0015] Trp, Tyr, Val.

[0016] Cysteine ​​is located at the C-terminus or one residue before the C-terminal residue and can be used for bioconjugation with polymers.

[0017] In another aspect, the invention features a method of bioconjugating a recombinant spider silk protein to a polyethylene glycol polymer, the method comprising the steps of:

[0018] a. Reducing disulfide bonds with tris(2-carboxyethyl)phosphine (TCEP) to obtain free thiol groups; the reaction temperature ranges from 2 to 10°C, and the pH ranges from 6.0 to 9.0. Preferably, the reaction temperature ranges from 3 to 5°C, and the pH ranges from 7.2 to 7.5.

[0019] b. Quenching with 1,2-bis(2-azidoethoxy)ethane

[0020] c. Coupling with 2- to 8-arm polyethylene glycol maleimide (PEG-Mal)

[0021] d. Formation of recombinant spider silk protein bioconjugates.

[0022] The assembled polymers obtained were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0023] The obtained bioconjugated recombinant spider silk protein contains 244 to 623 amino acid residues and is represented by the formula (NT-(Rep) x -C) y -PEG-Mal definition, wherein:

[0024] The N-terminal domain, NT, consists of 130 to 156 amino acid residues derived from the N-terminal domain of spider silk proteins;

[0025] Repeat domain (Rep) x , consisting of 87 to 463 amino acid residues derived from repeating sequences in spider silk proteins, where x is the number of repeating sequences from 1 to 8;

[0026] Domain C, which is a Cys(Z) residue, wherein Z is optional and, if present, is selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, The, Trp, Tyr, Val;

[0027] y is bioconjugated NT-(Rep) from 1 to 8 x -The number of C proteins;

[0028] PEG-Mal is 2- to 8-arm polyethylene glycol maleimide;

[0029] and optical isomers thereof.

[0030] In one embodiment, the bioconjugated recombinant spider silk protein is a recombinant spider silk protein having the general formula (NT-(Rep) x -C) y -PEG-Mal compounds, and have the following specific structures I-III:

[0031]

[0032] in

[0033] R1 = pentaerythritol nucleus;

[0034] R2 = tripentaerythritol nucleus;

[0035] n is an integer ranging from 50 to 400, corresponding to an average molecular weight ranging from 5000 Da to 10000 Da;

[0036] x is the number of repeating sequences from 1 to 8;

[0037] Domain C is a Cys(Z) residue, wherein Z is optional and, if present, is selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, The, Trp, Tyr, Val;

[0038] y is bioconjugated NT-(Rep) from 1 to 8 x -The number of C proteins;

[0039] and optical isomers thereof.

[0040] In another aspect, the invention features the use of recombinant spider silk protein bioconjugates to generate fibers in a biomimetic manner, which can be used in regenerative medicine, or as a cell culture scaffold.

[0041] In one embodiment, the invention features fibers obtained from the chemically modified recombinant spider silk protein derivatives described herein.

[0042] In another aspect, the invention features compounds obtainable by a synthetic method described herein or a method comprising a synthetic method described herein.

[0043] In another aspect, the invention features compounds obtained by a synthetic method described herein or a method comprising a synthetic method described herein.

[0044] In another aspect, the invention features novel intermediates described herein that are suitable for use in the synthetic methods described herein.

[0045] As will be appreciated by those skilled in the art, features and preferred embodiments of one aspect of the invention may also relate to other aspects of the invention. DETAILED DESCRIPTION

[0046] Recombinant spider silk proteins containing the NT domain have been identified as promising biopolymers that can be expressed in high yields in bacterial culture, have high solubility, and allow the spinning of artificial spider silk fibers without the use of organic solvents (Andersson et al., Nat. Chem. Biol. 2017, 13, 262-264).

[0047] The general formula NT-(Rep) x -C(NT-(Rep) x -Cys or NT-(Rep) x-CysAla) was bioconjugated with 2- to 8-arm polyethylene glycol maleimide (PEG-Mal) to generate recombinant spider silk protein bioconjugates (NT-(Rep) x -C) y -PEG-Mal. Depending on the number of PEG-Mal arms attached, the products formed are dimers to octamers, which can be further polymerized by NT-mediated dimerization at low pH. Therefore, this method enables cross-linking of recombinant spider silk proteins to a variable extent by changing the bioconjugation reaction conditions.

[0048] According to the present invention, the results of fiber spinning experiments indicate that artificial spider silk fibers can be produced by extruding chemically modified recombinant spider silk proteins into a low pH solution.

[0049] combination

[0050] Each and every compatible combination of the embodiments described above is expressly disclosed herein, as if each and every combination were individually expressly recited.

[0051] Examples of specific embodiments

[0052] The following examples further illustrate the present invention but should not be construed as limiting the scope of the invention in any way.

[0053] As an embodiment of the present invention, a molecule having the general formula (NT-(Rep) x -C) y -PEG-Mal bioconjugates of the following recombinant spider silk proteins:

[0054]

[0055]

[0056] Protein expression and purification

[0057] Constructs F2M, F4M, F6M, F1F, F2M*, F1T consisted of a His-tag (MGKHHHHHHPMSDYDIPTT) and a tobacco etch virus (TEV) protease cleavage site (ENLYFQG), followed by the N-terminal domain from N. clavipes FlSp (IANSPFSNPNTAEAFARSFVSNIVSSGEFGAQGAEDFEDIIQSLIQAQSMGKGRHDTKAKAKAMQVALASSIAELVIAESSGGDVQRKTNVISNALRNALMSTTGSPNEEFVHEVQDLIQMLSQEQINEV), a repeat region from the E. australis MaSp sequence (GNSGRGQGGYGQGSGGNAAAAAAAAAAAAAAAGQGGQGGYGRQSQGAGSAAAAAAAAAAAAAA AAA GSGQGGYGGQGQGGYGQSGNS) or (GNSGRGQGGYGQGSGGNAAAAAAAAAAAAAAAGQGGQGGYGRQSQGAGSAAAAAAAAAAAAAAAAPGNSGRGQGGYGQGSGGNAAAAAAAAAAAAAAAGQGGQGGYGRQSQGAGSAAAAAAAAAAAAAAA AAA GSGQGGYGGQGQGGYGQSGNS) or (GNSGRGQGGYGQGSGGNAAAAAAAAAAAAAAAGQGGQGGYGRQSQGAGSAAAAAAAAAAAAAAAAPGNSGRGQGGYGQGSGGNAAAAAAAAAAAAAAAGQGGQGGYGRQSQGAGSAAAAAAAAAAAAAAAAPGNSGRGQGGYGQGSGGNAAAAAAAAAAAAAAAGQGGQGGYGRQSQGAGSAAAAAAAAAAAAAAAAPGNSGRGQGGYGQGSGGNAAAAAAAAAAAAAAAGQGGQGGYGRQSQGAGSAAAAAAAAAAAAAAA AAAGSGQGGYGGQGQGGYGQSGNS) or C.clavipes FlSp sequence (DTSGPGQYYRSSSSSGGGGGGQGGPVVTEGPGGAGPGGYGPGSGPGGYGPGSGPGGYGPGSGPGGYGPGSGPGGYGPGSGPGGYGPGGYGPGSGPGGYGPGGTG PGGSGPGGYGPGGSGPGGSGPGGYGPGGSGPGGFGPGGSGPGGYGPGGSGPGGAGPGGVGPGGFGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPG GAGGAGGAGGAGGSGGAGGSGGTTIIEDLDITIDGADGPITISEELTISGAGGSGPGGAGPGGVGPGGSGPGGVGPGGGSGPGGVGPGSGPGGVGPGGAGGPYGPGGSGPGGAGGAG GPGGAYGPGGSYGPGGSGGPGGAGGPYGPGGEGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGEGGPYGPGGSYGPGGAGGPYGPGGPYGPGGEGPGGAGGPYGPGGVGPGGSGPGG AAA ) or from the TuSp sequence (SAARSGAQSSSTTTTSTSGSQAASSSASQASASSFAQASSASLAASSSFSSAFSSANTLSALGNVAYQLGFNVANTLGLGNAAGLGAALSQAVSSVGVGASSGTYANAVSNAVGQFLAGQGILNGANAASLASSFASALSASAASVASSSAAQSASQSQAAASAFSRAASQSASQ AAA ) and a C-terminal cysteine ​​(C) or cysteine ​​alanine (CysAla). The underlined are additional amino acids from the Not1 restriction site.

[0058] The recombinant spider silk protein construct was cloned into the pET28a(+) plasmid. The plasmid was transformed into BL21(DE)3 E. coli competent cells by heat shock and then incubated overnight on Luria broth (LB) plates containing 50 μg / mL kanamycin (kan). From this, a single colony was inoculated into 50 mL LB medium containing 50 μg / mL kan and grown overnight at 25°C. The overnight culture was then diluted to OD 0.05 in 1 L LB medium containing antibiotics. 600The OD was 0.01 and poured into a 2.5 L flask with baffles and incubated at 37 °C. 600 When it reached 0.6, the temperature was lowered to 25°C and expression was induced with 0.05 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). After overnight expression (F4M and F6M constructs were expressed for only 4 h), cells were harvested by centrifugation at 7000×g for 15 min and stored at −20°C until further use.

[0059] The cell pellet was resuspended in 10 mL of immobilized metal affinity chromatography (IMAC) loading buffer (20 mM sodium phosphate, pH 7.2, 300 mM NaCl, 15 mM imidazole) per gram and lysed by ultrasound. The soluble cell lysate portion was centrifuged at 4 ° C, 30,000 × g for 40 min and filtered through a 0.22 μm pore size filter. The lysate was loaded onto a HisTrap HP column (Cytivia) and eluted with 20 mM sodium phosphate, pH 7.2, 300 mM NaCl and 350 mM imidazole. Purity was further improved by gel filtration using a 16 / 600 Superdex PG200 size exclusion column at 20 mM sodium phosphate, pH 7.2, 300 mM NaCl.

[0060] General Synthesis

[0061] Recombinant spider silk protein bioconjugates 1.1-1.15 were prepared according to Scheme 1. Recombinant spider silk protein was reduced and reacted with PEG-Mal to generate the product.

[0062] Solution 1

[0063]

[0064] Reagents and conditions: (1) TCEP, 4°C, 24h; (2) N3-TEG-N3, 4°C, 1h

[0065] Synthetic products 1.1-1.15, general method. Take the synthesis of 1.9-1.11 of F1F-2-8 arm as an example

[0066] To a solution of F1F protein (0.6 mM solution, 1 mL, 0.0006 mmol, 1 eq) in phosphate buffer (c = 20 mM, pH = 7.2) was added a TCEP solution (50 mM, 0.12 mL, 0.006 mmol, 10 eq) in phosphate buffer (c = 20 mM, pH = 7.2). The mixture was vortexed quickly and placed at 4°C for 24 hours. Thereafter, 1,2-bis(2-azidoethoxy)ethane (6 mg, 0.030 mmol, 5 eq) was added, and the mixture was placed for another hour. The corresponding PEG-Mal solution in phosphate buffer (c = 20 mM, pH = 7.2) was then added, and the mixture was analyzed by SDS-PAGE after about 1 hour.

[0067] Recombinant spider silk protein bioconjugate with PEG(maleimide)2 of MW 5,000Da. Reduced protein was treated with a solution of PEG(maleimide)2 of MW 5000Da (1.5mg, 0.0003mmol, 0.43eq) in phosphate buffer (c=20mM, pH=7.2, 0.130mL). Relative proportions: double conjugated PEG 44%, single conjugated PEG 39%, unreacted Flag 12%.

[0068] Recombinant spider silk protein bioconjugate with PEG(maleimide)4 of MW 10000Da. Reduced protein was treated with a solution of PEG(maleimide)4 of MW 10000Da (1.5mg, 0.0002mmol, 0.22eq) in phosphate buffer (c=20mM, pH=7.2, 0.130mL). Relative proportions: tetraconjugated PEG 13%, triconjugated PEG 18%, diconjugated PEG 19%, monoconjugated PEG 12%, unreacted Flag 38%.

[0069] Recombinant spider silk protein bioconjugate with PEG(maleimide)8 with a MW of 10000Da. Reduced protein was treated with a solution of PEG(maleimide)8 with a MW of 10000Da (0.75mg, 0.00008mmol, 0.11eq) in phosphate buffer (c=20mM, pH=7.2, 0.065mL). Relative proportions: multi-conjugated PEG (5-8 proteins per PEG) 33%, tetra-conjugated PEG 13%, tri-conjugated PEG 13%, di-conjugated PEG 10%, unreacted F1F 31%.

[0070] The following compounds were obtained by the above method:

[0071]

[0072]

[0073]

[0074]

[0075]

[0076] Fiber Spinning

[0077] The F1F spider silk protein was dialyzed in pH 7.2, 20 mM sodium phosphate buffer and concentrated to 300 mg / mL. The spinning solution was then injected into a coagulation buffer (pH 5, 500 mM sodium acetate, 200 mM NaCl) using a syringe pump and collected on a spinning machine as described by Andersson et al. in Nat. Chem. Biol. 2017, 13, 262-264.

Claims

1. A recombinant spider silk protein comprising 244 to 623 amino acid residues and having the formula NT-(Rep) x -C defines where: - N-terminal domain NT, consisting of 130 to 156 amino acid residues derived from the N-terminal domain of spider silk proteins; -Repeat domain (Rep) x , consisting of 87 to 463 amino acid residues derived from the repeating sequence in spider silk protein, where x is the number of repeating sequences from 1 to 8; - Domain C, which is a Cys(Z) residue, wherein Z is optional and, if present, is selected from the following group: Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, The, Trp, Tyr, Val. The recombinant spider silk protein according to claim 1 , wherein domain C is Cys. The recombinant spider silk protein according to claim 1 , wherein domain C is CysAla.

4. The recombinant spider silk protein according to any one of the preceding claims, wherein (Rep) x The -domain preferably consists of 174-463 amino acid residues.

5. A method for bioconjugation of recombinant spider silk protein, comprising the following steps: a) Reduction of disulfide bonds to obtain free thiol groups using tris(2-carboxyethyl)phosphine, wherein the reaction temperature ranges from 2 to 10° C. and the pH ranges from 6.0 to 9.

0. b) quenching the product obtained in step a) with 1,2-bis(2-azidoethoxy)ethane; c) coupling the product obtained in step b) with 2- to 8-arm polyethylene glycol maleimide (PEG-Mal); d) Formation of bioconjugated recombinant spider silk protein.

6. The method according to claim 5, wherein the pH in step a) is preferably in the range of 7.2 to 7.

5.

7. The method according to claim 5, wherein the temperature in step a) is preferably in the range of 3°C to 5°C.

8. A bioconjugated recombinant spider silk protein comprising 244 to 623 amino acid residues and having the formula (NT-(Rep) x -C) y -PEG-Mal definition, wherein: - N-terminal domain NT, consisting of 130 to 156 amino acid residues derived from the N-terminal domain of spider silk proteins; -Repeat domain (Rep) x , consisting of 87 to 463 amino acid residues derived from the repeating sequence in spider silk protein, where x is the number of repeating sequences from 1 to 8; - domain C, which is a Cys(Z) residue, wherein Z is optional and, if present, is selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, The, Trp, Tyr, Val; -y is bioconjugated NT-(Rep) from 1 to 8 x -The number of C proteins; -PEG-Mal is 2- to 8-arm polyethylene glycol maleimide; and optical isomers thereof.

9. The bioconjugated recombinant spider silk protein according to claim 8, having the structure: in: n is an integer selected from 50 to 400, corresponding to an average molecular weight between 5000 Da and 10000 Da; x is the number of repeating sequences from 1 to 8; C is a Cys(Z) residue, wherein Z is optional and, if present, is selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, The, Trp, Tyr, Val.

10. The bioconjugated recombinant spider silk protein according to claim 8, having the structure: in: R1 is a pentaerythritol nucleus; n is an integer selected from 50 to 400, corresponding to an average molecular weight of 5000 Da to 10000 Da; x is the number of repeating sequences from 1 to 8; C is a Cys(Z) residue, wherein Z is optional and, if present, is selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, The, Trp, Tyr, Val.

11. The bioconjugated recombinant spider silk protein according to claim 8, having the structure: R2 is a tripentaerythritol nucleus; n is an integer selected from 50 to 400, corresponding to an average molecular weight of 5000 Da to 10000 Da; x is the number of repeating sequences from 1 to 8; C is a Cys(Z) residue, wherein Z is optional and, if present, is selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, The, Trp, Tyr, Val.

12. Bioconjugated recombinant spider silk proteins for applications in regenerative medicine.

13. The bioconjugated recombinant spider silk protein according to claim 12, used as a cell culture scaffold.

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