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Fermenter Production of an Artificial Fab Fragment, Rationally Designed for the Antigen Cystatin, and Its Optimized Crystallization Through Constant Domain Shuffling

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Schiweck,  Wolfram
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Skerra,  Arne
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Citation

Schiweck, W., & Skerra, A. (1995). Fermenter Production of an Artificial Fab Fragment, Rationally Designed for the Antigen Cystatin, and Its Optimized Crystallization Through Constant Domain Shuffling. Proteins: Structure, Function, and Bioinformatics, 23(4), 561-565. doi:10.1002/prot.340230411.


Cite as: http://hdl.handle.net/21.11116/0000-0007-B38D-0
Abstract
The synthetic antibody model "M41" was rationally designed with a binding site complementary to chicken egg white cystatin as the prescribed antigen. In order to permit comparison between the computer model and an experimental three-dimensional structure of the artificial protein, its X-ray crystallographic analysis was pursued. For this purpose, M41 was expressed as a recombinant Fab fragment in E. coli by medium cell density fermentation employing the tightly regulated tetracycline promoter. The Fab fragment was efficiently purified via a His-6 tail fused to its heavy chain and immobilized metal affinity chromatography. To raise the chances for the productive formation of crystal packing contacts, three versions of the Fab fragment were generated with differing constant domains. One of these, the variant with murine CK and CH1γ1 domains, was successfully crystallized by microseeding in a sitting drop. The orthorhombic crystals exhibited symmetry of the space group P212121 with unit cell dimensions a = 104.7 A, b = 113.9 A, c = 98.8 A and diffracted X-rays to a nominal resolution of 2.5 A.