Anticalins
The invention relates to the production of novel proteins exhibiting binding 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 genetic engineering methods. Like immunoglobulin, the anticalin thus obtained can be used to identify or bind molecular structures.
1. An isolated lipocalin mutein 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 structurally 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 mutein is effective to bind a ligand with a determinable affinity of at least 10 5 M −1 .
2. The lipocalin mutein of claim 1 , wherein the lipocalin mutein is derived from a human lipocalin.
3. The lipocalin mutein of claim 2 , wherein the lipocalin mutein 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.
4. The lipocalin mutein of claim 2 , wherein the lipocalin mutein 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.
5. The lipocalin mutein of claim 1 , wherein at least one amino acid of each of said four peptide loops has been mutated.
6. The lipocalin mutein of claim 1 , wherein the lipocalin mutein is bound to a solid phase, and wherein the bound lipocalin mutein binds a ligand.
7. The lipocalin mutein of claim 1 , wherein the lipocalin mutein is fused to a fusion partner.
8. The lipocalin mutein of claim 7 , 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 a lipocalin mutein with different or the same ligand specificity.
9. The lipocalin mutein of claim 1 , wherein the lipocalin mutein is coupled to a further compound.
10. The lipocalin mutein of claim 9 , wherein the further compound is selected from the group consisting of a protein, a radioactive moiety and a molecule with a defined binding characteristic.
11. The lipocalin mutein of claim 10 , wherein the protein is selected from the group consisting of an enzyme, an antibody, and a lipocalin mutein with different or the same ligand specificity.