Cloning, expression and characterisation of a single-chain Fv antibody fragment against domoic acid in Escherichia coli.
Domoic acid is a potent neuroexcitatory toxin that causes amnesic shellfish poisoning in humans through ingestion of contaminated shellfish. The variable regions of the heavy chain (V(H)) and light chain (V(L)) of an antibody specific for domoic acid were cloned from a mouse hybridoma cell line and used to construct single-chain antibody fragments (scFvs) in a variety of formats. V(H)-linker-V(L) scFvs were expressed better in Escherichia coli than the V(L)-linker-V(H) format, while use of the commonly used (Gly4Ser)3 inter-domain linker resulted in higher yields than a longer (Gly4Ser)6 linker variant. Higher soluble protein yields were achieved in E. coli TOP 10 than in E. coli XL1-Blue cells and co-production of the E. coli disulfide bond isomerase enzyme DsbC allowed higher cell densities to be attained during scFv production, leading to increased yields of recombinant protein. The purified scFv exhibited binding similar to the parent monoclonal antibody and is being used to develop an immunosensor to detect domoic acid in contaminated shellfish samples.
Hu
Xuejun
X
Department of Chemical and Environmental Sciences, University of Limerick, Plassey Technology Park, Limerick, Ireland.
O'Dwyer
Ronan
R
Wall
J Gerard
JG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D007608:Kainic Acid / Q000031:analogs & derivatives* / Q000276:immunology; D051379:Mice; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; C012301:domoic acid; D007608:Kainic Acid