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 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 K(D) of 30.2(+/-3.6) nM. The derivative compound digitoxigenin is bound even more tightly, with a K(D) 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.
Schlehuber
S
S
Lehrstuhl für Biologische Chemie, Technische Universität München, Freising-Weihenstephan, D-85350, Germany.
Beste
G
G
Skerra
A
A
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004076:Digoxigenin / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005453:Fluorescence; D014644:Genetic Variation / Q000235:genetics; D019476:Insect Proteins; D008024:Ligands; D054835:Lipocalin 1; D054834:Lipocalins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D019151:Peptide Library; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D013816:Thermodynamics; D017186:Titrimetry
D002352:Carrier Proteins; C575984:DigA16; D019476:Insect Proteins; D008024:Ligands; D054835:Lipocalin 1; D054834:Lipocalins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C053849:bilin-binding protein, insect; D000469:Alkaline Phosphatase; D004076:Digoxigenin