TY - JOUR
T1 - Synthesis and characterization of a functional intact IgG in a prokaryotic cell-free expression system
AU - Frey, Stephan
AU - Haslbeck, Martin
AU - Hainzl, Otmar
AU - Buchner, Johannes
N1 - Funding Information:
Authentic MAK33, CK-MM and DsbC were kindly provided by Helmut Lenz and Erhard Fernholz, respectively (Roche Diagnostics, Penzberg, Germany). The Grp94 plasmid was prepared by Rene Leubert. We gratefully acknowledge the experimental help of Tatiana Krebs, Kerstin Mayer and Isabella Moeckel and thank Matthias Feige, Moritz Marcinowki and Emma Simpson for reading the manuscript. This work was funded by grants from Deutsche Forschungsgesellschaft and Fonds der Chemischen Insustrie to J.B.
PY - 2008/1/1
Y1 - 2008/1/1
N2 - 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.
AB - 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.
KW - Antibody stability
KW - Cell-free expression system
KW - Escherichia coli lysate
KW - Glycosylation
KW - IgG1
KW - MAK33
KW - Protein disulfide isomerase
UR - http://www.scopus.com/inward/record.url?scp=37349010476&partnerID=8YFLogxK
U2 - 10.1515/BC.2008.007
DO - 10.1515/BC.2008.007
M3 - Article
C2 - 18095868
AN - SCOPUS:37349010476
SN - 1431-6730
VL - 389
SP - 37
EP - 45
JO - Biological Chemistry
JF - Biological Chemistry
IS - 1
ER -