PMID: 2120697Jul 1, 1990Paper

Expression of mouse immunoglobulin light and heavy chain variable regions in Escherichia coli and reconstitution of antigen-binding activity

Protein Engineering
H FieldA R Rees

Abstract

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 expressi...Continue Reading

Citations

Feb 10, 1994·Journal of Immunological Methods·R J Owens, R J Young
Oct 1, 1992·Molecular Immunology·J AnthonyS C Ng
Jan 1, 1994·Biotechnology Advances·B E Fischer
Apr 1, 1993·Current Opinion in Immunology·A Skerra
Aug 1, 1993·Australian and New Zealand Journal of Medicine·R L Ward, N J Hawkins
May 15, 1996·Annals of the New York Academy of Sciences·J S Harrison, E Keshavarz-Moore
Dec 1, 1992·Immunological Reviews·J R Adair
Jan 1, 1993·Cell Biophysics·J S HustonH Oppermann

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