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S. Schlehuber, A. Skerra
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 ligandbinding 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 socalled 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 bilinbinding 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 Streptag method. The major fraction of the purified protein appeared to possess the proper pattern of altogether four disulphide bonds. The ligandbinding 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 ligandbinding 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.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 382, 10/2001
Pages: 1335 - 1342