Patent ID: 7250297

Claim:
A method of producing a lipocalin that binds to a ligand, the lipocalin having a cylindrical β-pleated sheet supersecondary structural region comprising eight β-strands connected pair-wise by four loops at one end to define thereby a binding pocket, said four loops corresponding to those segments which comprise amino acids 28-45, 58-69, 86-99, and 114-129 of the linear polypeptide sequence of the bilin binding protein of Pieris brassicae SEQ ID NO: 20, wherein at least one amino acid of each of at least three of said four loops has been mutated and wherein said lipocalin is effective to bind said ligand with a determinable affinity of at least 10 5 M −1 , said method comprising: providing a plurality of nucleic acid molecules coding for a lipocalin and mutating at least some of the nucleic acid molecules such that at least one amino acid of each of at least three of said four loops has been mutated to form a library of lipocalin nucleic acid molecules; operably fusing at least part of the library of lipocalin nucleic acid molecules at the 3′-end with a gene coding for at least part of a coat protein pIII of a filamentous bacteriophage of the M13-family, thereby forming a library of lipocalin fusion genes; expressing a plurality of the lipocalin fusion genes on the surface of a plurality of bacteriophages, respectively, to form a library of lipocalin phages; isolating at least one of the lipocalin phages by binding the at least one lipocalin phage to the ligand; preparing the lipocalin fusion gene from the lipocalin phage; and expressing in a host cell at least part of the lipocalin fusion gene to obtain thereby at least one lipocalin.