Duocalins: engineered ligand-binding proteins with dual specificity derived from the lipocalin fold.

Anticalins comprise a novel class of receptor proteins with predetermined ligand specificities which were engineered using the lipocalin fold. Attractive features of these artificial ligand-binding proteins include their small size and monomeric nature, being composed of a single polypeptide chain. Here we report the construction of a functional fusion protein from two independent anticalins, a so-called duocalin. The gene for the fusion protein was assembled from nucleotide sequences encoding an anticalin with fluorescein specificity on the one hand and an anticalin with digoxigenin specificity on the other. Both engineered lipocalins were previously selected from a random library prepared on the basis of the bilin-binding protein, a natural lipocalin abundant in insects. The corresponding fusion protein was expressed in a secretable form in E. coli cells and isolated from the periplasmic fraction using the Strep-tag method. The major fraction of the purified protein appeared to possess the proper pattern of altogether four disulphide bonds. The ligand-binding behaviour of the fusion protein was investigated both by solid phase ELISA and in fluorescence titration experiments. Our results demonstrate that the novel fusion protein has retained both ligand specificities. Up to now, dimerized ligand-binding proteins were mostly derived from recombinant antibody fragments. Compared with those constructs the duocalins, either with bispecific or with bivalent target recognition properties, should provide useful reagents for various purposes in biotechnology.