Chemically modified artificial spider silk protein

Abstract

The present invention aims to avoid the requirements for both terminal domains in the gene construct of recombinant spidroin and still produce fibers such as spider silk in a biomimetic manner. **Solution**: The present invention relates to recombinant spidroin bioconjugates (chemically modified artificial spider silk proteins). More specifically, the present invention relates to recombinant spidroin bioconjugates with polyethylene glycol polymers and their use in regenerative medicine.

Description

Technical Field

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

Background Art

[0002] Spider silk is made of protein (spidroin) and generally consists of three structural subunits, a non-repetitive N-terminal domain and C-terminal domain (NT and CT), and a long central region with a highly repetitive sequence (REP). NT and CT are involved in the silk formation process, and the central region determines the mechanical properties of silk fibers.

[0003] Recombinant spidroin can be produced using heterologous expression in bacteria, yeast, and other expression systems. Recombinant spidroin usually has fewer repetitive sequences (Rep) in the central repetitive region and often lacks one or both of the terminal domains, unlike natural spidroin.

[0004] The structural transition of spidroin from a soluble dope to solid fibers is mediated by changes in the pH and ionic composition of the aqueous environment and shear force. Studies of the terminal domains have shown that lowering the pH to about 5.5 causes NT to dimerize and stabilize, thereby strongly binding spidroin to each other, while CT unfolds to form amyloid-like fibrils and may function as a nucleation species to promote the conversion of the repetitive region to a beta-sheet structure (Andersson et al. PLoS Biol. 2014, 12, e1001921).

[0005] Recombinant spidroins lacking one or both of the terminal domains cannot reproduce the molecular mechanism of native silk spinning, especially the structural transition of the terminal domain. Therefore, non-native methods such as coagulation are required for the conversion to the beta-sheet structure. To obtain artificial spider silk by a biomimetic method, the recombinant spidroin must contain both terminal domains and accurately reproduce the conditions in the spider silk gland to ensure the assembly of the recombinant spidroin.

[0006] A recombinant spidroin that contains both terminal domains and can polymerize in a biomimetic manner to form spider silk-like fibers, and a method for producing such fibers are described in EP3263593.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Non-Patent Documents

[0008]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0009] The present invention aims to avoid the requirements for both terminal domains in the gene construct of recombinant spidroin and still produce fibers such as spider silk by a biomimetic method.

Means for Solving the Problems

[0010] In a first aspect, the present invention relates to a recombinant spidroin consisting of 244 to 620 amino acid residues and defined by the formula NT-(Rep)x -C, which consists of three characteristics: a. The N-terminal domain NT consists of a fragment of 130 to 156 amino acid residues derived from the N-terminal domain of spidroin; b. The repetitive domain (Rep)x consists of 87 to 463 amino acid residues derived from the repetitive sequence in spidroin; where x is the number of repetitive sequences; the (Rep)x-domain preferably consists of 174 to 463 amino acid residues; c. Domain C is a Cys(Z) residue, where Z is optional, and if present, it is selected from: Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, The, Trp, Tyr, Val. Cysteine is located at the C-terminus or one residue before the C-terminus and can be used for bioconjugation to the polymer.

[0011] In another aspect, the present invention features a method for bioconjugating a recombinant spidroin to a polyethylene glycol polymer, comprising the following steps: a. Reducing the disulfide bond using tris(2-carboxyethyl)phosphine (TCEP) to obtain free thiol groups; the reaction temperature is in the range of 2 to 10 °C and the pH is in the range of 6.0 to 9.0. Preferably, the reaction temperature is in the range of 3 to 5 °C and the pH is in the range of 7.2 to 7.5. b. Quenching with 1,2-bis(2-azidoethoxy)ethane c. Coupling with polyethylene glycol maleimide (PEG-Mal) having 2 to 8 arms d. Formation of the recombinant spidroin bioconjugate

[0012] The obtained assembled polymer is analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0013] The obtained bioconjugated recombinant spidroin consists of 244 to 623 amino acid residues and is defined by the formula: NT-(Rep)x-C)y-PEG-Mal: (Here, NT represents an N-terminal domain consisting of 130 to 156 amino acid residues derived from the N-terminal domain of spidroin, (Rep) represents a repetitive domain consisting of 87 to 463 amino acid residues derived from the repetitive sequence in spidroin, x is the number of repetitive sequences from 1 to 8, C is the domain of Cys(Z) residue, Z is optional, and when 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 and Val; y is the number of bioconjugated NT-(Rep)x-C proteins from 1 to 8, PEG-Mal is a 2- to 8-armed polyethylene glycol maleimide.) And its optical isomers.

[0014] In one embodiment, the bioconjugated recombinant spidroin is a compound having the general formula (NT-(Rep)x-C)y-PEG-Mal and has the following specific structures I to III:

[0015]

Chemical formula

[0016]

Chemical formula

[0017]

Chemical formula

[0018] (Here, R1 represents a pentaerythritol core, R2 represents a tripentaerythritol core, n is an integer in the range of 50 to 400 and corresponds to an average molecular weight between 5000 Da and 10000 Da, x represents the number of repeating sequences from 1 to 8, C is the domain of the Cys(Z) residue, where 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, y is the number of bioconjugate NT-(Rep)x-C proteins from 1 to 8) and its optical isomers.

[0019] In another aspect, the present invention features the use of recombinant spidroin bioconjugates for the biomimetic production of fibers that can be used as scaffolds for regenerative medicine and cell culture.

[0020] In one embodiment, the present invention features fibers obtained from the chemically modified recombinant spidroin derivatives described herein.

[0021] In another aspect, the present invention features a compound obtained by the synthetic method described herein, or a method comprising the synthetic method described herein.

[0022] In another aspect, the present invention features a compound obtained by the synthetic method described herein, or a method comprising the synthetic method described herein.

[0023] In another aspect, the present invention features a novel intermediate suitable for use in the synthetic methods described herein, as described herein.

[0024] As will be understood by those skilled in the art, the features and preferred embodiments of one aspect of the present invention are also relevant to other aspects of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0025] Recombinant spidroin containing an NT domain has been identified as a promising biopolymer that can be expressed in high yields in bacterial culture, has high solubility, and can spin artificial spider silk fibers without using organic solvents (Andersson et al., Nat. Chem. Biol. 2017, 13, 262-264).

[0026] Using the bioconjugation of recombinant spidroin having the general formula NT-(Rep)x-C(NT-(Rep)x-Cys or NT-(Rep)x-CysAla) with 2- to 8-arm polyethylene glycol maleimide (PEG-Mal), a recombinant spidroin bioconjugate (NT-(Rep)x-C)y-PEG-Mal was generated. The formed product is a dimer to octamer linked according to the number of arms of PEG-Mal and can be further polymerized by dimerization via NT at low pH. Thus, this method can change the degree of crosslinking of recombinant spidroin by changing the bioconjugation reaction conditions.

[0027] According to the present invention, it was demonstrated from the results of fiber spinning experiments that artificial spider silk fibers can be produced by extruding chemically modified recombinant spidroin into a low pH solution.

[0028] Combination Each compatible combination of the above-described embodiments is explicitly disclosed herein as if each combination were individually and explicitly described.

Example

[0029] The following examples further illustrate the present invention but should not be construed as limiting the scope of the present invention in any way.

[0030] As an example of the present invention, the following recombinant spidroin bioconjugates having the general formula (NT-(Rep)x-C)y-PEG-Mal were prepared.

[0031]

Table 1

[0032]

Table 2

[0033]

Table 3

[0034] Protein Expression and Purification F2M, F4M, F6M, F1F, F2M*, F1T are the FlSp (IANSPFSNPNTAEAFARSFVSNIVSSGEFGAQGAEDFEDIIQSLIQAQSMGKGRHDTKAKAKAMQVALASSIAELVIAESSGGDVQRKTNVISNALRNALMSTTGSPNEEFVHEVQDLIQMLSQEQINEV) of N. clavipes, the E. australis MaSp sequence (GNSGRGQGGYGQGSGGNAAAAAAAAAAAA AAA GQGGQGGYGRQSQGAGSAAAAAAAAAAAAAA AAA GSGQGGYGGQGQGGYGQSGNS) or (GNSGRGQGGYGQGSGGNAAAAAAAAAAAAAGQGGQGGYGRQSQGAGSAAAAAAAAAAAAPGNSGRGQGGYGQGSGGNAAAAAAAAAAAAAGQGGGYGRQSQGAGSAAAAAAAAAAAAAAAA AAA(GSGQGGQGQYGQSGNS) or (GNSGRGQGGYGQGSGGNAAAAAAAAAAAAAGQGGQGGYGRQSQGAGSAAAAAAAAAAAAAAPGNSGRGQGGYGQGSGGNAAAAAAAAAAAAAGQGGGYGRQSQGAGSAAAAAAAAAAAAAAAAPGNSGRGQGGYGQGSGGNAAAAAAAAAAAAAAAAAAAGQGGYGRQSQGAGSAAAAAAAAAAAAAAA AAA (GSGQQGGYGQSGNS) or the C. clavipes FlSp sequence (DTSGPGQYYRSSSSGGGGGGQGGPVVTEGPGGAGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGYGPGGSGPGGYGPGGTGPGGSGPGGYGPGGSGPGGSGPGGYGPGGSGPGGFGPGGSGPGGYGPGGSGPGGAGPGGVGPGGFGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGGAGGAGGAGGSGGAGGSGGTTIIEDLDITIDGADGPITISEELTISGAGGSGPGGAGPGGVGPGGSGPGGVGPGGSGPGGVGPGGSGPGGVGPGGAGGPYGPGGSGPGGAGGAGGPGGAYGPGGSYGPGGSGGPGGAGGPYGPGGEGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGEGGPYGPGGSYGPGGAGGPYGPGGPYGPGGEGPGGAGGPYGPGGVGPGGSGPGG AAA ) or the TuSp sequence (SARSGAQSSTTSSGSQAASSQASASQASSFAAAAAASSFSATLSALGNVAYQLGFNVANLGLGNAAAGLGAASQAVSGVGGASGTYANAVSNAVGQFLAGQGILNGANAASLASSFASALSASAASVASAAQSASQSQAAAASAFSRAQSASQ AAA ) obtained from, and a C-terminal cysteine (C) or cysteine alanine (CysAla). The underline is the additional amino acid derived from the Not1 restriction site.

[0035] The recombinant spidroin construct was cloned into the pET28a(+) plasmid. Using this plasmid, BL21(DE)3 E. coli competent cells were transformed by heat shock and subsequently cultured overnight on Luria broth (LB) plates containing 50 μg / mL kanamycin (kan). From there, a single colony was inoculated into 50 mL of LB medium containing 50 μg / mL of kan and cultured overnight at 25 °C. The overnight culture was diluted with 1 L of LB medium containing antibiotics to an OD 600 of 0.01, poured into a 2.5 L baffled flask, and cultured at 37 °C. When the OD 600 reached 0.6, the temperature was lowered to 25 °C and expression was induced using 0.05 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). After overnight expression (F4M and F6M constructs were expressed for only 4 hours), the cells were harvested by centrifugation at 7000×g for 15 minutes and stored at -20 °C until further use.

[0036] The cell pellet was suspended 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 sonication. The soluble cell lysate fraction was separated by centrifugation at 30,000×g for 40 minutes at 4 °C and filtered through a 0.22 μm pore size filter. This lysate was applied to a HisTrap HP column (Cytivia) and eluted using 20 mM sodium phosphate, pH 7.2, 300 mM NaCl, 350 mM imidazole. The purity was further improved by gel filtration using a 16 / 600 Superdex PG200 size exclusion column in 20 mM sodium phosphate, pH 7.2, 300 mM NaCl.

[0037] General Synthesis Recombinant spidroin bioconjugates 1.1 - 1.15 were prepared according to Scheme 1. The recombinant spidroin was reduced and reacted with PEG-Mal to obtain a product.

[0038]

Chemical formula

[0039] Synthesis of products 1.1 - 1.15, general method. Synthesis example of F1F - 2 - 8arm 1.9 - 1.11

[0040] 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), a TCEP solution (50 mM, 0.12 mL, 0.006 mmol, 10 eq) in phosphate buffer (c = 20 mM, pH = 7.2) was added. The mixture was quickly stirred and left at 4 °C for 24 hours. Then, 1,2 - bis(2 - azidoethoxy)ethane (6 mg, 0.030 mmol, 5 eq) was added and the mixture was left standing for another 1 hour. Then, the corresponding PEG - Mal solution in phosphate buffer (c = 20 mM, pH = 7.2) was added and the mixture was analyzed by SDS - PAGE approximately 1 hour later.

[0041] Recombinant spidroin bioconjugation with PEG(Maleimide)2 of MW 5000Da

[0042] The reduced protein was treated with a solution of PEG(maleimide)2 of MW 5000 Da (1.5 mg, 0.0003 mmol, 0.43 eq) in phosphate buffer (c = 20 mM, pH = 7.2, 0.130 mL). Relative ratio: bis - conjugated PEG 44%, mono - conjugated PEG 39%, unreacted Flag 12%.

[0043] Recombinant spidroin bioconjugation with PEG (Maleimide)4 of MW 10000 Da

[0044] The reduced protein was treated with a solution of PEG(Maleimide)4 with a molecular weight of 10000 Da (1.5 mg, 0.0002 mmol, 0.22 eq) in phosphate buffer (c = 20 mM, pH = 7.2, 0.130 mL). Relative ratio: tetra-conjugated PEG 13%, tri-conjugated PEG 18%, bis-conjugated PEG 19%, mono-conjugated PEG 12%, unreacted Flag 38%.

[0045] Recombinant Spidroin Bioconjugation with PEG(Maleimide)4 of Molecular Weight 10000 Da

[0046] The reduced protein was treated with a solution of PEG(Maleimide)8 with a molecular weight of 10000 Da (0.75 mg, 0.00008 mmol, 0.11 eq) in phosphate buffer (c = 20 mM, pH = 7.2, 0.065 mL). Relative ratio: poly-conjugated PEG (5 - 8 proteins per PEG) 33%, tetra-conjugated PEG 13%, tri-conjugated PEG 13%, bis-conjugated PEG 10%, unreacted F1F 31%.

[0047] By the above method, the following compounds were obtained.

[0048]

Table 4

[0049]

Table 5

[0050]

Table 6

[0051] Fiber Spinning

[0052] The F1F spidroin was dialyzed against 20 mM sodium phosphate buffer at pH 7.2 and concentrated to 300 mg / mL. This dope was injected into the coagulation buffer (pH 5, 500 mM sodium acetate, 200 mM NaCl) using a syringe pump and collected on a spinning frame using a syringe pump as described by Andersson et al., Nat. Chem. Biol. 2017, 13, 262-264.

Claims

1. Consisting of 244 to 623 amino acid residues, of the formula: NT-(Rep)x-C where NT represents an N-terminal domain consisting of 130 to 156 amino acid residues derived from the N-terminal domain of spidroin, (Rep) represents a repetitive domain consisting of 87 to 463 amino acid residues derived from the repetitive sequence in spidroin, x is the number of repetitive sequences from 1 to 8, C is the domain of the Cys(Z) residue, Z is optional, and when present, is selected from the group Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, The, Trp, Tyr and Val), a recombinant spidroin defined by.

2. The recombinant spidroin according to claim 1, wherein the domain C is Cys.

3. The recombinant spidroin according to claim 1, wherein the domain C is CysAla.

4. (Rep)x is preferably a domain consisting of 174 to 463 amino acid residues, the recombinant spidroin according to any of the preceding claims.

5. A method for bioconjugation of recombinant spidroin, comprising the following steps: a) Reducing the disulfide bond with tris(2-carboxyethyl)phosphine to obtain free thiol groups (where 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) Forming a bioconjugation of recombinant spidroin.

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. Consisting of 244 to 623 amino acid residues, of the formula: NT-(Rep)x-C)y-PEG-Mal where NT represents an N-terminal domain consisting of 130 to 156 amino acid residues derived from the N-terminal domain of spidroin, (Rep) represents a repetitive domain consisting of 87 to 463 amino acid residues derived from the repetitive sequence of piderin, and x is the number of repetitive sequences from 1 to 8, C is the domain of Cys(Z) residue, Z is optional, and when present, is selected from Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, The, Trp, Tyr, and Val; y is the number of bioconjugate NT-(Rep)x-C proteins from 1 to 8; PEG-Mal is 2- to 8-arm polyethylene glycol maleimide), a bioconjugate recombinant spidroin and its optical isomers defined thereby.

9. The bioconjugate recombinant spidroin according to claim 8 having the structure of the following formula (wherein n is an integer from 50 to 400, corresponding to an average molecular weight between 5000 Da and 10000 Da, x represents the number of repetitive sequences from 1 to 8, C is the domain of Cys(Z) residue, Z is optional, and when present, is selected from the group of Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, The, Trp, Tyr, and Val). 【Chemical 1】

10. The bioconjugate recombinant spidroin according to claim 8 having the structure of the following formula (wherein R1 represents a pentaerythritol core, n is an integer from 50 to 400, corresponding to an average molecular weight between 5000 Da and 10000 Da, x represents the number of repetitive sequences from 1 to 8, C is the domain of Cys(Z) residue, Z is optional, and when present, is selected from the group of Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, The, Trp, Tyr, and Val). [Chemical Formula 2]

11. The bioconjugate recombinant spidroin according to claim 8 having the structure of the following formula (wherein R2 is a tripentaerythritol core, n is an integer from 50 to 400, corresponding to an average molecular weight between 5000 Da and 10000 Da, x represents the number of repetitive sequences from 1 to 8, C is a domain of the Cys(Z) residue, where 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, and Val). [Chemical Formula 3] Claim 12 A bioconjugate recombinant spidroin for use in regenerative medicine. Claim 13 The bioconjugate recombinant spidroin according to claim 12 for use as a cell culture scaffold.

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