Chemically modified engineered spider silk proteins
AssigneeLatvian Institute of Organic Synthesis
InventorsKristaps Jaudzems, Viktors Romanuks, Jekabs Fridmanis, Anna Lina Bula, Gints Smits
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Abstract
The present invention relates to recombinant spidroin bioconjugates (chemically modified engineered spider silk proteins). More particularly, the invention relates to recombinant spidroin bioconjugates with poly-ethyleneglycol polymers and their use in regenerative medicine.
Claims13
A recombinant spidroin comprising from 244 to 623 amino acid residues and defined by the formula NT-(Rep)x-C wherein:
- an N-terminal domain NT consisting of 130 to156 amino acid residues derived from the N-terminal domain of a spidroin;
- a repetitive domain (Rep)x consisting of from 87 to 463 amino acid residues derived from the repetitive sequences in a spidroin, wherein x is a number of repetitive sequences from 1 to 8;
- a domain C is Cys (Z) residue, where Z is optional, if 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, Val.
A method of bioconjugation of the recombinant spidroin comprising the steps of:
- a) reducing the disulfide bond to obtain free thiol group with tris(2-carboxyethyl) phosphine, wherein the reaction temperature is in range from 2 to 10° C. and pH is in the range from 6.0 to 9.0.
- b) quenching of obtained product 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 the bioconjugated recombinant spidroin.
A bioconjugated recombinant spidroin comprising from 244 to 623 amino acid residues and defined by the formula (NT-(Rep)x-C)y-PEG-Mal wherein:
- an N-terminal domain NT consisting of 130 to 156 amino acid residues derived from the N-terminal domain of a spidroin;
- a repetitive domain (Rep) consisting of 87 to 463 amino acid residues derived from the repetitive sequences in a spidroin, wherein x is a number of repetitive sequences from 1 to 8;
- a domain C is Cys (Z) residue, where Z is optional, if 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, Val;
- y is a number of bioconjugated NT-(Rep)x-C proteins from 1 to 8;
- PEG-Mal is 2- to 8-arm polyethylene glycol maleimide;
- and optical isomers thereof.
A bioconjugated recombinant spidroins for use in regenerative medicine.
Dependencies are inferred from the claim text.