Bacterial expression and purification of recombinant bovine Fab fragments.
We have previously described a recombinant phagemid expression vector, pComBov, designed for the production of native sequence bovine monoclonal antibodies (mAb) generated by antibody phage display. Bovine mAb Fab fragments isolated from libraries constructed using pComBov in Escherichia coli strain XL1-Blue, which is routinely used for antibodies expressed on the surface of phage, were expressed at very low yields. Therefore, a study was undertaken to determine optimal growth conditions for maximal expression of bovine Fab fragments in E. coli. By varying the E. coli strain, and the temperature and length of the culture growth, we were able to substantially increase the yield of soluble Fab fragments. A high yield of Fab fragments was found in the culture growth medium, which enabled us to devise a rapid and simple single-step method for the purification of native (nondenatured) Fabs based on immobilized metal affinity chromatography against a six-histidine amino acid carboxyl-terminal extension of the heavy-chain constant region. Using these methods we were able to express and purify antigen-specific bovine Fab fragments from E. coli.
O'Brien
Philippa M
PM
Department of Veterinary Pathology, University of Glasgow, Bearsden, Glasgow, Scotland, United Kingdom. p.obrien@vet.gla.ac.uk
Maxwell
Gavin
G
Campo
M Saveria
MS
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006639:Histidine; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D013696:Temperature
D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D006639:Histidine