Optimisation of production of a domoic acid-binding scFv antibody fragment in Escherichia coli using molecular chaperones and functional immobilisation on a mesoporous silicate support.
Domoic acid is a potent neurotoxin that can lead to amnesic shellfish poisoning in humans through ingestion of contaminated shellfish. We have produced and purified an anti-domoic acid single-chain Fragment variable (scFv) antibody fragment from the Escherichia coli periplasm. Yields of functional protein were increased by up to 100-fold upon co-production of E. coli DnaKJE molecular chaperones but co-overproduction of GroESL led to a reduction in solubility of the scFv. Co-production of the peptidyl-prolyl isomerase trigger factor resulted in accumulation of unprocessed scFv in the E. coli cytoplasm. This was due to an apparent bottleneck in translocation of the cytoplasmic membrane by the recombinant polypeptide. Co-expression of the E. coli disulfide bond isomerase dsbC increased scFv yields by delaying lysis of the host bacterial cells though this effect was not synergistic with molecular chaperone co-production. Meanwhile, use of a cold-shock promoter for protein production led to accumulation of greater amounts of scFv polypeptide which was predominantly in insoluble form and could not be rescued by chaperones. Purification of the scFv was achieved using an optimised metal affinity chromatography procedure and the purified protein bound domoic acid when immobilised on a mesoporous silicate support. The work outlines the potential benefit of applying a molecular chaperone/folding catalyst screening approach to improve antibody fragment production for applications such as sensor development.
Hu
Xuejun
X
Department of Chemical and Environmental Sciences, University of Limerick, Plassey Technology Park, Limerick, Ireland.
O'Hara
Liam
L
White
Simon
S
Magner
Edmond
E
Kane
Marian
M
Wall
J Gerard
JG
D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005838:Genotype; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D007608:Kainic Acid / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D009498:Neurotoxins / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D015183:Restriction Mapping
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D018832:Molecular Chaperones; D009498:Neurotoxins; D011994:Recombinant Proteins; C012301:domoic acid; D007608:Kainic Acid