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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred


IMPACT FACTOR 2018: 3.014
5-year IMPACT FACTOR: 3.162

CiteScore 2018: 3.09

SCImago Journal Rank (SJR) 2018: 1.482
Source Normalized Impact per Paper (SNIP) 2018: 0.820

Online
ISSN
1437-4315
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Volume 389, Issue 1

Issues

Synthesis and characterization of a functional intact IgG in a prokaryotic cell-free expression system

Stephan Frey
  • 1Center for Integrated Protein Science Munich (CIPSM) and Department of Chemistry, Technical University of Munich, Lichtenbergstraße 4, D-85747 Garching, Germany
  • Other articles by this author:
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/ Martin Haslbeck
  • 2Center for Integrated Protein Science Munich (CIPSM) and Department of Chemistry, Technical University of Munich, Lichtenbergstraße 4, D-85747 Garching, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Otmar Hainzl
  • 3Center for Integrated Protein Science Munich (CIPSM) and Department of Chemistry, Technical University of Munich, Lichtenbergstraße 4, D-85747 Garching, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Johannes Buchner
  • 4Center for Integrated Protein Science Munich (CIPSM) and Department of Chemistry, Technical University of Munich, Lichtenbergstraße 4, D-85747 Garching, Germany
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  • De Gruyter OnlineGoogle Scholar
Published Online: 2007-12-20 | DOI: https://doi.org/10.1515/BC.2008.007

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.

Keywords: antibody stability; cell-free expression system; Escherichia coli lysate; glycosylation; IgG1; MAK33; protein disulfide isomerase

About the article

Corresponding author


Received: 2007-05-31

Accepted: 2007-09-24

Published Online: 2007-12-20

Published in Print: 2008-01-01


Citation Information: Biological Chemistry, Volume 389, Issue 1, Pages 37–45, ISSN (Online) 14374315, ISSN (Print) 14316730, DOI: https://doi.org/10.1515/BC.2008.007.

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