Method for identifying hetero-multimeric modified ubiquitin proteins with binding capability to ligands

The method of anyone of the preceding claims, wherein said modified monomeric ubiquitin protein is obtained by genetically engineering of DNA encoding for ubiquitin, and expressing of said protein in prokaryotic or eukaryotic organisms or in vitro.

The method of anyone of the preceding claims, wherein said multimeric protein is provided by an in vitro selection method which is preferably a display method, optionally a phage display, ribosomal display, TAT phage display, yeast display, bacterial display, cell surface display or mRNA display method.

The method of anyone of the preceding claims, wherein said ligand is an antigen or hapten.

The method of anyone of the preceding claims, wherein further 1 to 7 additional amino acids are substituted in at least one of the monomeric ubiquitin proteins, which are optionally selected from one or more of the amino acids in positions 36, 44, 70, 71, and optionally additionally 62, 63, and 64 or 72 and 73 or 8 of SEQ ID NO:

• 1.

The method of anyone of the preceding claims, wherein said population of hetero-multimeric fusion proteins of ubiquitin is provided by genetically fusing two DNA libraries encoding each for differently modified monomeric proteins, translating the DNA into hetero-multimeric fusion proteins, displaying said proteins and screening the displayed proteins on the presence of modified hetero-multimeric ubiquitin proteins comprising monomeric ubiquitin proteins being linked together in a head-to-tail arrangement wherein said modified hetero-multimeric ubiquitin proteins bind to said ligand with a specific binding affinity of Kd in a range of 10−7-10−12M and exhibit a monovalent binding activity with respect to said ligand or wherein said population of hetero-multimeric fusion proteins of ubiquitin is provided by chemical synthesis of the proteins.

A DNA library containing DNA encoding for a population of hetero-multimeric ubiquitin fusion proteins originating from monomeric ubiquitins, each multimeric protein comprising two or more modified ubiquitin monomers linked together in a head-to-tail arrangement wherein at least two of each of said monomers of said multimeric protein are 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% or at least 90% or at least 95% to the unmodified protein.