Synthesis and characterization of a functional intact IgG in a prokaryotic cell-free expression system
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Stephan Frey
Abstract
Antibodies are an important component of the immune system of higher eukaryotes. Furthermore, they are effective tools in basic research, medical diagnostics and therapy. Recombinant expression of these heterotetrameric, disulfide-bridged proteins is usually performed in mammalian cells. Here, we describe the cell-free expression of a mouse monoclonal antibody, MAK33, in a coupled transcription/translation system, based on an Escherichia coli lysate. Both the heavy and the light chain can be produced efficiently in this setup. However, they fail to form functional antibodies. With a view to overcome folding and oxidation defects, we supplemented the system with the oxidoreductases PDI (protein disulfide isomerase) and DsbC and the ER-specific chaperones Grp94 and BiP; furthermore, we optimized the redox conditions. We found that functional antibodies can only be obtained in the presence of an oxidoreductase. In contrast, the addition of Grp94 and/or BiP had no influence on the productive folding reaction. The comparison of the antibody expressed in vitro with MAK33 expressed in cell culture showed that the in vitro expressed antibody is correctly assembled, disulfide-bridged and shows identical antigen affinity. The stability of the in vitro expressed non-glycosylated IgG is comparable to that of the authentic antibody.
©2008 by Walter de Gruyter Berlin New York
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- Differential effects of novel tumour-derived p53 mutations on the transformation of NIH-3T3 cells
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- Heparinase selectively sheds heparan sulphate from the endothelial glycocalyx
- Inhibition of human μ-calpain by conformationally constrained calpastatin peptides
- Cloning, expression and characterization of insulin-degrading enzyme from tomato (Solanum lycopersicum)
