A novel type of receptor protein, based on the lipocalin scaffold, with specificity for digoxigenin
We demonstrate that the bilin-binding protein, a member of the lipocalin family of proteins, can be structurally reshaped in order to specifically complex digoxigenin, a steroid ligand commonly used for the non-radioactive labelling of biomolecules. 16 amino acid residues, distributed across the fou...
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Veröffentlicht in: | Journal of molecular biology 2000-04, Vol.297 (5), p.1105-1120 |
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Sprache: | eng |
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Zusammenfassung: | We demonstrate that the bilin-binding protein, a member of the lipocalin family of proteins, can be structurally reshaped in order to specifically complex digoxigenin, a steroid ligand commonly used for the non-radioactive labelling of biomolecules. 16 amino acid residues, distributed across the four loops which form the binding site of the bilin-binding protein, were subjected to targeted random mutagenesis. From the resulting library the variant DigA16 was obtained by combined use of phage display and a filter-sandwich colony screening assay, followed by in vitro affinity maturation. DigA16 possesses strong binding activity and high specificity for the digoxigenin group, with a KD of 30.2(±3.6) nM. The derivative compound digitoxigenin is bound even more tightly, with a KD of 2.0(±0.52) nM, whereas the steroid glycoside ouabain is not recognized at all. Fusion proteins between DigA16 and alkaline phosphatase were constructed and shown to retain both the digoxigenin-binding function and enzymatic activity, irrespective of whether the enzyme was fused to the N or the C terminus of the bilin-binding protein variant. Our findings suggest that the lipocalin scaffold can be generally employed for the construction of specific receptor proteins, so-called “anticalins”, which provide a promising alternative to recombinant antibody fragments. |
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ISSN: | 0022-2836 1089-8638 |
DOI: | 10.1006/jmbi.2000.3646 |