Anticalins
The invention relates to the production of novel proteins exhibiting bonding activity for certain ligands, the so-called anticalins. To this end, the structure of peptides of the lipocalin family is modified by amino acid replacement in their natural ligand binding pocket using generic engineering methods. Alike immunoglobulin, the anticalin thus obtained can be used to identify or bond molecular structures.
1. An isolated lipocalin having a cylindrical β-pleated sheet supersecondary structural region comprising eight β-strands connected pair-wise by four peptide loops at one end to define thereby a binding pocket, said four peptide 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 peptide loops has been mutated and wherein said lipocalin is effective to bind a ligand selected from the group consisting of a polypeptide and a peptide with a determinable affinity of at least 10 5 M −1 .
2. The lipocalin of claim 1 , wherein at least one amino acid of each of said four peptide loops has been mutated.
3. The lipocalin of claim 1 , wherein the lipocalin is a human lipocalin.
4. The lipocalin of claim 3 , wherein the lipocalin is derived from any one of the group consisting of, human retinol-binding protein, and human apolipoprotein D.
5. The lipocalin of claim 1 , wherein the mutated amino acids in the region of the four peptide loops correspond to sequence positions 34-37, 58, 60, 69, 88, 90, 93, 95, 97, 114, 116, 125 and 127 of the linear polypeptide sequence of bilin-binding protein SEQ ID NO: 20.
6. The lipocalin of claim 4 , wherein said peptide loops have been mutated at one or more positions selected from the group consisting of amino acids 34, 35, 36, 37, 58, 60, 69, 88, 90, 93, 95, 97, 114, 116, 125 and 127 of said bilin-binding protein, wherein said amino acid position number is based on SEQ ID NO: 20.
7. The lipocalin of claim 1 , wherein the lipocalin is derived from human apolipoprotein D and the mutated amino acids in the region of the four peptide loops correspond to sequence positions 34-37, 59, 61, 70, 87, 89, 92, 94, 96, 113, 115, 123 and 125 of the linear polypeptide sequence of human apolipoprotein D as set forth in SEQ ID NO: 19.
8. The lipocalin of claim 1 , wherein the lipocalin is derived from human retinol-binding protein and the mutated amino acids in the region of the four peptide loops correspond to sequence positions 32-35, 57, 59, 73, 90, 92, 95, 100, 102, 119, 121, 131, and 133 of the linear polypeptide sequence of human retinol binding protein as set forth in SEQ ID NO: 18.
9. The lipocalin of claim 1 , wherein the ligand is a tumor-specific cellular surface molecule.
10. The lipocalin of claim 1 , wherein the lipocalin is bound to a solid phase, and wherein the bound lipocalin binds a ligand.
11. The lipocalin of claim 1 , wherein the lipocalin is fused to a fusion partner.
12. The lipocalin of claim 11 , wherein the fusion partner is selected from the group consisting of the albumin-binding domain of protein G, protein A, an antibody fragment, an oligomerizing domain, a toxin and an lipocalin with different or the same ligand specificity.
13. The lipocalin of claim 1 , wherein the lipocalin is coupled to a further compound.
14. The lipocalin of claim 13 , wherein the further compound is selected from the group consisting of a protein, a radioactive moiety and a molecule with a defined binding characteristic.
15. The lipocalin of claim 14 , wherein the protein is selected from the group consisting of an enzyme, an antibody, and a lipocalin with different or the same ligand specificity.