Duocalins: Engineered Ligand-Binding Proteins with Dual Specificity Derived from the Lipocalin Fold
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Abstract
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.
Copyright © 2001 by Walter de Gruyter GmbH & Co. KG
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Articles in the same Issue
- Highlight: Evolution in Vivo, in Vitro and in Machina
- Modeling Genetic Networks and Their Evolution: A Complex Dynamical Systems Perspective
- Evolution in Silico and in Vitro: The RNA Model
- Divergent Evolution of (??)8-Barrel Enzymes
- RNA-Catalyzed Carbon-Carbon Bond Formation
- Toward Automated Nucleic Acid Enzyme Selection
- Duocalins: Engineered Ligand-Binding Proteins with Dual Specificity Derived from the Lipocalin Fold
- The Stochastic Evolution of Catalysts in Spatially Resolved Molecular Systems
- Fragment-Based Flexible Ligand Docking by Evolutionary Optimization
- Specific Nucleoprotein Complexes within Adenovirus Capsids
- ERH (Enhancer of Rudimentary Homologue), a Conserved Factor Identical between Frog and Human, Is a Transcriptional Repressor
- Signal Transduction by the Chemokine Receptor CXCR5: Structural Requirements for G Protein Activation Analyzed by Chimeric CXCR1/CXCR5 Molecules
- Arginine-Specific Cysteine Proteinase from Porphyromonas gingivalis as a Convenient Tool in Protein Chemistry
- Chemokine-Induced Secretion of Gelatinase B in Primary Human Monocytes
