Affilin-novel binding molecules based on human gamma-B-crystallin, an all beta-sheet protein |
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Authors: | Ebersbach Hilmar Fiedler Erik Scheuermann Tanja Fiedler Markus Stubbs Milton T Reimann Carola Proetzel Gabriele Rudolph Rainer Fiedler Ulrike |
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Affiliation: | Scil Proteins GmbH, Heinrich Damerow Str. 1, 06120 Halle (Saale), Germany. hebersbach@bioc.unizh.ch |
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Abstract: | The concept of novel binding proteins as an alternative to antibodies has undergone rapid development and is now ready for practical use in a wide range of applications. Alternative binding proteins, based on suitable scaffolds with desirable properties, are selected from combinatorial libraries in vitro. Here, we describe an approach using a beta-sheet of human gamma-B-crystallin to generate a universal binding site through randomization of eight solvent-exposed amino acid residues selected according to structural and sequence analyses. Specific variants, so-called Affilin, have been isolated from a phage display library against a variety of targets that differ considerably in size and structure. The isolated Affilin variants can be produced in Escherichia coli as soluble proteins and have a high level of thermodynamic stability. The crystal structures of the human wild-type gamma-B-crystallin and a selected Affilin variant have been determined to 1.7 A and 2.0 A resolution, respectively. Comparison of the two molecules indicates that the human gamma-B-crystallin tolerates amino acid exchanges with no major structural change. We conclude that the intrinsically stable and easily expressed gamma-B-crystallin provides a suitable framework for the generation of novel binding molecules. |
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Keywords: | CDR, complementarity-determining region GdmHCl, guanidinium hydrochloride IMAC, immobilized metal-affinity chromatography MIA, melanoma inhibitory activity NGF, nerve growth factor proNGF, pro-form of NGF SPC-x-xx, named Affilin molecule SPR, surface plasmon resonance TMB, 3, 3′, 5′, 5′-tetramethylbenzidine |
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