Expression of mouse immunoglobulin light and heavy chain variable regions in Escherichia coli and reconstitution of antigen-binding activity.
The expression of immunoglobulin heavy and light chain variable regions in the cytoplasm of Escherichia coli and formation of a functional heterodimer has been demonstrated. Variable domain sequences were taken from the heavy and light chain cDNAs of the monoclonal antibody Gloop 2 and engineered for expression in a dual origin expression vector. The engineered genes vhg2 and vlg2 were separately subcloned into the vector, creating two expression plasmids. Expression of the heavy and light chain variable region genes (encoding 116 and 109 amino acids respectively) was investigated in eight E. coli strains; the polypeptides were rapidly degraded in a host strain optimized for expression and in E. coli strains deficient in the major protease La (lon-). Accumulation was permitted in severely protease-deficient E. coli having a defective heat-shock response. A lon- mutation in this genetic background permitted even higher accumulation. Expression levels were 7 and 1% of total bacterial protein for light and heavy chain variable regions respectively. Expression of the heavy chain variable region gene was increased by including a longer Shine-Dalgarno sequence. Similar constructions in the light chain vector had no effect on expression levels. The insoluble variable region polypeptides were reconstituted into a heterodimer possessing the full antigen binding characteristics of both the parent monoclonal antibody and its Fab fragment.
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Laboratory of Molecular Biophysics, University of Oxford, UK.
Yarranton
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Rees
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D000818:Animals; D001666:Binding Sites, Antibody; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D051379:Mice; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic
D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region