PMID:9653035
| Citation |
Martineau, P, Jones, P and Winter, G (1998) Expression of an antibody fragment at high levels in the bacterial cytoplasm. J. Mol. Biol. 280:117-27 |
|---|---|
| Abstract |
Recombinant antibody fragments expressed in the cytoplasm of cells have considerable practical potential. However in the reducing environment of the cytoplasm, the intradomain disulphide bonds are not formed and the fragments are unstable and expressed in low yields. Here we attempted to overcome these limitations. We first isolated an antibody single chain Fv fragment that binds and activates an inactive mutant beta-galactosidase. We then subjected the gene encoding the scFv fragment to random mutation in vitro by error-prone polymerase chain reaction, and co-expressed the mutant beta-galactosidase and mutant antibody fragments in lac- bacteria. By plating on limiting lactose, we selected for antibody mutants with improved expression, and after four successive rounds of mutation and selection, isolated an antibody fragment that is expressed in the bacterial cytoplasm with yields of 0.5 g/l in a shaker flask (A600 nm of 5.5) and 3.1 g/l (A600 nm=33) in a fermentor. Analysis of the mutant antibody fragments revealed that the disulphide bonds are reduced in the cytoplasm, and that the fragments could be denatured and renatured efficiently under reducing conditions in vitro. This shows that with a suitable method of screening or selection, it is possible to make folded and functional antibody fragments in excellent yield in the cytoplasm. |
| Links |
PubMed Online version:10.1006/jmbi.1998.1840 |
| Keywords |
Amino Acid Sequence; Bacteriophage T7/genetics; Cytoplasm/metabolism; Disulfides; Enzyme Activation; Escherichia coli/metabolism; Gene Expression; Humans; Immunoglobulin Fragments/genetics; Immunoglobulin Fragments/immunology; Immunoglobulin Variable Region/genetics; Immunoglobulin Variable Region/immunology; Molecular Sequence Data; Mutagenesis; Promoter Regions, Genetic; Protein Folding; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/immunology; beta-Galactosidase/immunology |
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