PMID:12801908
| Citation |
Zheng, L, Baumann, U and Reymond, JL (2003) Production of a functional catalytic antibody ScFv-NusA fusion protein in bacterial cytoplasm. J. Biochem. 133:577-81 |
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| Abstract |
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. |
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| Keywords |
Antibodies, Bacterial/biosynthesis; Antibodies, Bacterial/genetics; Antibodies, Bacterial/metabolism; Antibodies, Catalytic/biosynthesis; Antibodies, Catalytic/genetics; Antibodies, Catalytic/metabolism; Bacteriophage T7/genetics; Cytoplasm/metabolism; Disulfides/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins; Ether, Ethyl/metabolism; Immunoglobulin Fragments/biosynthesis; Immunoglobulin Fragments/genetics; Immunoglobulin Fragments/metabolism; Peptide Elongation Factors/biosynthesis; Peptide Elongation Factors/genetics; Peptide Elongation Factors/metabolism; Recombinant Fusion Proteins/biosynthesis; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/metabolism; Stereoisomerism; Transcription Factors/biosynthesis; Transcription Factors/genetics; Transcription Factors/metabolism |
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