Patent ID: 7947475

Claim:
A method for producing a modified variant BBPI in a bacterial cell, said method comprising: a) mutating a polynucleotide that encodes an unmodified precursor variant BBPI to generate a polynucleotide construct encoding a modified variant BBPI, wherein the unmodified precursor variant BBPI comprises a BBPI scaffold selected from the group consisting of: BBI (SEQ ID NO: 13), BBIt (SEQ ID NO:185), BBI-AV (SEQ ID NO:186), BBIt-AV (SEQ ID NO:187), BBIt-VEGK (SEQ ID NO:640), BBIt-VEGT (SEQ ID NO:641), BBIt-VEGKD (SEQ ID NO:642), BBdb (SEQ ID NO:449), BBsb3 (SEQ ID NO:450), BBtc (SEQ ID NO:451), BBdb-AV (SEQ ID NO:452), BBsb3-AV (SEQ ID NO:453) and BBtc-AV (SEQ ID NO:454), wherein the second protease inhibitory loop of the BBPI scaffold is replaced with a binding peptide chosen from a vascular endothelial growth factor (VEGF) binding peptide, a fibroblast growth factor-5 (FGF-5) binding peptide, a transforming growth factor β (TGF β) binding peptide and a tumor necrosis factor α (TNF α) binding peptide, and wherein the mutation in step (a) results in an amino acid substitution in the backbone of the unmodified precursor variant at least at one amino acid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of SEQ ID NO: 187, wherein the substitution at position 1 is to A or C, the substitution at position 4 is to V, the substitution at position 5 is to P or A, the substitution at position 11 is to G, the substitution at position 13 is to Y, I, F, M, L, V, K or R, the substitution at position 18 is to I, V or L, the substitution at position 25 is to K, N, W, I, A, R or L, the substitution at position 27 is to R, K, V, A or Q, the substitution at position 29 is to R, K, P or E, the substitution at position 31 is to Q, H, E, A, R, W, K or T, the substitution at position 38 is to N, K or R, the substitution at position 40 is to H, K, Q, R or Y, the substitution at position 50 is to R, Q, K, T, V, M or S, the substitution at position 52 is to K, T, R, Q, L, H, A, M, S or E, the substitution at position 55 is to M and the substitution at position 65 is to E, Q or D; wherein the modified variant BBPI comprises the unmodified precursor variant BBPI with said amino acid substitution in the backbone; and b) introducing said polynucleotide construct encoding a modified variant BBPI into a bacterial host cell; c) culturing said bacterial cell under suitable culture conditions to allow expression of said modified variant BBPI; and d) producing said modified variant BBPI, wherein said modified variant BBPI has a greater trypsin inhibitory activity than the corresponding unmodified precursor variant BBPI.