Synthesis and characterization of a functional intact IgG in a prokaryotic cell-free expression system.
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.
Frey
Stephan
S
Center for Integrated Protein Science Munich and Department of Chemistry, Technical University of Munich, D-85747 Garching, Germany.
Haslbeck
Martin
M
Hainzl
Otmar
O
Buchner
Johannes
J
D000915:Antibody Affinity / Q000276:immunology; D015153:Blotting, Western; D002474:Cell-Free System; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004220:Disulfides / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000096:biosynthesis; D018832:Molecular Chaperones / Q000378:metabolism; D010084:Oxidation-Reduction; D010957:Plasmids / Q000235:genetics; D011387:Prokaryotic Cells / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000096:biosynthesis / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance
D004220:Disulfides; D018840:HSP70 Heat-Shock Proteins; D007074:Immunoglobulin G; D008565:Membrane Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; C018551:glucose-regulated proteins; D019704:Protein Disulfide-Isomerases