Spider silk fusion protein structures for binding to an organic target

A method for providing a protein structure capable of selective interaction with an organic target, wherein said protein structure is a polymer comprising as a repeating structural unit a recombinant fusion protein that is capable of selective interaction with the organic target and comprising moieties B, REP CT, and optionally NT, wherein the polymer comprises more than 100 fusion protein structural units, and wherein:

• B is a non-spidroin moiety of more than 30 amino acid residues, which provides the capacity of selective interaction with the organic target,
• wherein the B moiety is selected from the group consisting of the Z domain of staphylococcal protein A, staphylococcal protein A and the E, D, A, B and C domains thereof;
• streptococcal protein G, the albumin-binding domain thereof and the C1, C2 and C3 domains thereof;
• streptavidin and monomeric streptavidin (M4);
• GA modules from Finegoldia magna;
• and variants thereof wherein the B moiety is capable of selective interaction with the organic target;
• REP is a moiety of from 70 to 300 amino acid residues and is derived from the repetitive fragment of a spider silk protein,
• wherein the REP moiety is selected from the group of L(AG)nL, L(AG)nAL, L(GA)nL, L(GA)nGL, wherein
• n is an integer from 2 to 10;
• each individual A segment is an amino acid sequence of from 8 to 18 amino acid residues, wherein from 0 to 3 of the amino acid residues are not Ala, and the remaining amino acid residues are Ala;
• each individual G segment is an amino acid sequence of from 12 to 30 amino acid residues, wherein at least 40% of the amino acid residues are Gly;
• and
• each individual L segment is a linker amino acid sequence of from 0 to 20 amino acid residues;
• CT is a moiety of from 70 to 120 amino acid residues and is derived from the C-terminal fragment of a spider silk protein, wherein the CT moiety has at least 50% identity to SEQ ID NO:
• 9 or at least 80% identity to SEQ ID NO:
• 7;
• and
• NT, when optionally present, is a moiety of from 100 to 160 amino acid residues and is derived from the N-terminal fragment of a spider silk protein;
• wherein the NT moiety has at least 50% identity to SEQ ID NO:
• 8 or at least 80% identity to SEQ ID NO:
• 6,
• said method comprising the steps of:
• (a) providing said recombinant fusion protein;
• and
• (b) subjecting the fusion protein to conditions to achieve formation of a polymer comprising the recombinant fusion protein.

An affinity medium for immobilization of an organic target, said affinity medium comprising a fusion protein that is capable of selective interaction with the organic target and comprising the moieties B, REP, CT, and optionally NT, wherein:

• B is a non-spidroin moiety of more than 30 amino acid residues, which provides the capacity of selective interaction with the organic target,
• wherein the B moiety is selected from the group consisting of the Z domain of staphylococcal protein A, staphylococcal protein A and the E, D, A, B and C domains thereof;
• streptococcal protein G, the albumin-binding domain thereof and the C1, C2 and C3 domains thereof;
• streptavidin and monomeric streptavidin (M4);
• GA modules from Finegoldia magna;
• and variants thereof wherein the B moiety is capable of selective interaction with the organic target;
• REP is a moiety of from 70 to 300 amino acid residues and is derived from the repetitive fragment of a spider silk protein,
• wherein the REP moiety is selected from the group of L(AG)nL, L(AG)nAL, L(GA)nL, L(GA)nGL, wherein
• n is an integer from 2 to 10;
• each individual A segment is an amino acid sequence of from 8 to 18 amino acid residues, wherein from 0 to 3 of the amino acid residues are not Ala, and the remaining amino acid residues are Ala;
• each individual G segment is an amino acid sequence of from 12 to 30 amino acid residues, wherein at least 40% of the amino acid residues are Gly;
• and
• each individual L segment is a linker amino acid sequence of from 0 to 20 amino acid residues;
• CT is a moiety of from 70 to 120 amino acid residues and is derived from the C-terminal fragment of a spider silk protein, wherein the CT moiety has at least 50% identity to SEQ ID NO:
• 9 or at least 80% identity to SEQ ID NO:
• 7;
• and
• NT, when optionally present, is a moiety of from 100 to 160 amino acid residues and is derived from the N-terminal fragment of a spider silk protein;
• wherein the NT moiety has at least 50% identity to SEQ ID NO:
• 8 or at least 80% identity to SEQ ID NO:
• 6.