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US 9,493,523 B2Granted
RNF8-FHA domain-modified protein and method of producing the same
AssigneeGeneFrontier Corporation
InventorsKanehisa Kojoh, Shizue Katoh, Akira Miyakoshi, Mikiko Nakamura
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Abstract
Provided is an antigen-binding protein prepared merely by a method of in vitro selection using the RNF8-FHA domain, which has no intramolecular disulfide bond and functions in cells as it is. One to four loops extending from the FHA domain are randomized, and a recognition site for a target molecule is artificially created on the FHA domain surface to construct an RNF8-FHA domain library. Using the library, an antigen-binding protein is efficiently selected in vitro.
Claims8
A method of producing an artificial antibody against a predetermined antigen, comprising:
- (i) preparing a RNF8-FHA domain library comprising randomized-loop polypeptides obtained by randomizing two or more of the following loops (a′) to (d′) in the E3 ubiquitin ligase RNF8-FHA domain comprising the amino acid sequence of SEQ ID NO:1:
- (a′) a loop consisting of the 41st-46th amino acid residues of the RNF8 protein shown by SEQ ID NO:1,
- (b′) a loop consisting of the 49th-62nd amino acid residues of the RNF8 protein shown by SEQ ID NO:1,
- (c′) a loop consisting of the 78th-83rd amino acid residues of the RNF8 protein shown by SEQ ID NO:1,
- (d′) a loop consisting of the 108th-118th amino acid residues of the RNF8 protein shown by SEQ ID NO:1,
- wherein each of the randomized-loop polypeptides comprises the amino acid sequence shown by SEQ ID NO:1 except that a random sequence is introduced into two or more loops selected from the group consisting of (a′) to (d′), and no amino acid residues are deleted from, substituted for, inserted into, or added to any regions of the amino acid sequence other than loops (a′), (b′), (c′), and (d′), and
- (ii) selecting a randomized-loop polypeptide that binds to the antigen as the artificial antibody from among the randomized-loop polypeptides of the RNF8-FHA domain polypeptide library,
- wherein unmodified RNF8-FHA domain has substantially no binding affinity for the antigen.
5 more elements
Dependencies are inferred from the claim text.