Patent ID: 8748351

Claim:
A method for identifying a hetero-multimeric modified ubiquitin with binding capability to a ligand with a binding affinity, with respect to the ligand, that did not exist previously, comprising the following steps: a) providing a population of hetero-multimeric modified ubiquitin originating from monomeric modified ubiquitin proteins, said population comprising hetero-multimeric proteins comprising two or more ubiquitin monomers linked together in a head-to-tail arrangement wherein at least one of said monomers of said hetero-multimeric protein is differently modified at least by substitutions of surface exposed amino acids in at least three amino acids located in positions 2, 4, 6, 8, 62, 63, 64, 65, 66, and 68 of SEQ ID NO: 1, said modified monomeric protein having an amino acid sequence identity of at least 80%, at least 90%, or at least 95% to the unmodified ubiquitin protein; b) providing a potential ligand to said population of differently modified proteins; c) contacting said population of differently modified proteins with said ligand; d) identifying a hetero-multimeric modified protein by a screening process, wherein said modified hetero-multimeric protein binds to said ligand with a specific binding affinity of Kd in a range of 10 −7 -10 −12 M and exhibits a monovalent binding activity with respect to said ligand; and optionally e) isolating said hetero-multimeric modified ubiquitin with said binding affinity, wherein a hetero-multimeric modified ubiquitin that binds to a ligand with a binding affinity, with respect to the ligand, that did not exist previously is identified.