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

1. 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.

2. The method of claim 1 , wherein said hetero-multimeric protein is a hetero-dimeric or hetero-trimeric protein.

3. The method of claim 1 , wherein said modified monomeric protein further comprises one or more insertions of 1 to 10 amino acids and/or one or more deletions of 1 to 7 amino acids as compared to SEQ ID NO: 1, further optionally wherein said modified monomeric ubiquitin protein comprises at least 6 and at most 14 substitutions of amino acids as compared to SEQ ID NO: 1,

and further wherein as compared to SEQ ID NO: 1, said modified hetero-dimeric ubiquitin protein comprises:

(i) at least 12 and at most 28 substitutions; and/or

(ii) at least 1 and at most 20 insertions; and/or

(iii) at least 1 and at most 14 deletions.

4. The method of claim 1 , 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.

5. The method of claim 1 , wherein said screening process is a display method, optionally selected from the group consisting of a phage display method, a ribosomal display method, a TAT phage display method, a yeast display method, a bacterial display method, a cell surface display method, or an mRNA display method.

6. The method of claim 1 , wherein said ligand is an antigen or hapten.

7. The method of claim 1 , wherein a further 1 to 7 additional amino acids are substituted in at least one of the monomeric ubiquitin proteins, which further 1 to 7 additional amino acids 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.

8. The method of claim 1 , wherein said population of hetero-multimeric fusion proteins of ubiquitin is provided by genetically fusing two DNA libraries each encoding differently modified monomeric proteins, translating the DNA into hetero-multimeric fusion proteins, displaying said proteins and screening the displayed proteins for 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 −12 M 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.

9. 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:

(i) 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;

(ii) the multimeric protein binds to a ligand with a binding affinity, with respect to the ligand, that did not exist previously; and

(iii) said modified monomeric protein having has an amino acid sequence identity of at least 80%, at least 90%, or at least 95% to SEQ ID NO: 1.

10. A protein library obtained by expression of the DNA library of claim 9 .

11. A prokaryotic or eukaryotic cell or a population of phages containing the DNA or protein library according to claim 9 or 10 .

12. A polynucleotide encoding for a fusion protein of said protein library of claim 10 .

13. A vector comprising a polynucleotide according to claim 12 .