Fibronectin type III repeat based protein scaffolds with alternative binding surfaces

摘要

Protein scaffolds and scaffold libraries based on a fibronectin type III (FN3) repeat with an alternative binding surface design, isolated nucleic acids encoding the protein scaffolds, vectors, host cells, and methods of making thereof are useful in the generation of therapeutic molecules and treatment and diagnosis of diseases and disorders.

主权项

1. A method of making a library of fibronectin module of type III (FN3) domains having a diversified C-CD-F-FG alternative surface formed by a C beta-strand, a CD loop, an F beta-strand, and an FG loop, comprising
a. providing a reference FN3 domain polypeptide having the amino acid sequence at least 80% identical to that of SEQ ID NO: 27; b. introducing diversity into the reference FN3 domain polypeptide by mutating at least one C beta-strand residue and at least one F beta-strand residue to form the FN3 domain library having the diversified C-CD-F-FG alternative surface, wherein the at least one residue in the C beta-strand is mutated with the proviso that S30 is not mutated (residue numbering according to SEQ ID NO: 27), wherein the at least one residue in the F beta-strand is mutated with the proviso that E66 is not mutated (residue numbering according to SEQ ID NO: 27), wherein each of the C beta-strand residues L32, Q34 and Q36 are mutated (residue numbering according to SEQ ID NO: 27) or each of the F-beta strand residues T68, S70 and Y72 are mutated (residue numbering according to SEQ ID NO: 27).