Production of a functional catalytic antibody ScFv-NusA fusion protein in bacterial cytoplasm.
Functional expression of catalytic antibodies in the cytoplasm of E. coli is potentially of great interest in searching for new catalysts by genetic selection. Herein, a catalytic antibody single chain Fv (ScFv) 14D9, which catalyzes a highly enantioselective protonation, was expressed as a NusA fusion protein under the T7 promoter. A functional disulfide-containing ScFv fusion protein was obtained in the oxidizing environment of bacterial cytoplasm. The 14D9 ScFv could not be overexpressed alone without NusA fusion. The highly soluble NusA protein most likely retards aggregate formation of ScFv and indirectly supports correct folding and disulfide bridge formation in the fusion construct ScFv-NusA. The ScFv-NusA fusion product shows highly enantioselective, specific, hapten inhibited catalytic activity comparable to its parent monoclonal antibody, 14D9. The NusA fusion method might be generally helpful for functional antibody expression in vivo and for the new development of biocatalysts by genetic selection.
Zheng
Lei
L
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.
Baumann
Ulrich
U
Reymond
Jean-Louis
JL
D000907:Antibodies, Bacterial / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D017151:Antibodies, Catalytic / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D017123:Bacteriophage T7 / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D004986:Ether / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D010445:Peptide Elongation Factors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D035602:Transcriptional Elongation Factors
D000907:Antibodies, Bacterial; D017151:Antibodies, Catalytic; D004220:Disulfides; D029968:Escherichia coli Proteins; D007128:Immunoglobulin Fragments; D010445:Peptide Elongation Factors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; D004986:Ether