Article: article from journal or magazin.
"Peptabody": a new type of high avidity binding protein.
Proceedings of the National Academy of Sciences of the United States of America
Publication types: Journal Article Publication Status: ppublish
A new type of high avidity binding molecule, termed "peptabody" was created by harnessing the effect of multivalent interaction. A short peptide ligand was fused via a semi-rigid hinge region with the coiled-coil assembly domain of the cartilage oligomeric matrix protein, resulting in a pentameric multivalent binding molecule. In the first peptabody (Pab-S) described here, a peptide (S) specific for the mouse B-cell lymphoma BCL1 surface Ig idiotype, was selected from a phage display library. A fusion gene was constructed encoding peptide S, followed by the 24 aa hinge region from camel IgG and a modified 55 aa cartilage oligomeric matrix protein pentamerization domain. The Pab-S fusion protein was expressed in Escherichia coli in a soluble form at high levels and purified in a single step by metal-affinity chromatography. Pab-S specifically bound the BCL1 surface idiotype with an avidity of about 1 nM, which corresponds to a 2 x 10(5)-fold increase compared with the affinity of the synthetic peptide S itself. Biochemical characterization showed that Pab-S is a stable homopentamer of about 85 kDa, with interchain disulfide bonds. Pab-S can be dissociated under denaturing and reducing conditions and reassociated as a pentamer with full-binding activity. This intrinsic feature provides an easy way to combine Pab molecules with two different peptide specificities, thus producing heteropentamers with bispecific and/or chelating properties.
Animals, Binding, Competitive, Blotting, Western, Chromatography, Gel, Computer Simulation, Escherichia coli/genetics, Extracellular Matrix Proteins, Glycoproteins/chemistry, Glycoproteins/metabolism, Ligands, Lymphoma, B-Cell, Mice, Mice, Inbred BALB C, Models, Molecular, Peptides/chemistry, Peptides/genetics, Plasmids, Protein Binding, Protein Conformation, Receptors, Cell Surface/metabolism, Recombinant Fusion Proteins/chemistry, Recombinant Fusion Proteins/metabolism, Sequence Analysis, Signal Transduction, Tumor Cells, Cultured
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