Bacterial expression and in vitro refolding of a single-chain fv antibody specific for human plasma apolipoprotein B-100.
From the cloned heavy and light chains of a murine monoclonal antibody (mAbB23) which is specific for human apolipoprotein (apo) B-100 of plasma low-density lipoproteins, a vector was designed for expression of a single-chain antibody (scFv) of mAbB23 in Escherichia coli. The expression vector was constructed so that the scFv gene (V(L)-linker-V(H)) was expressed under the control of the T7 promoter. The inclusion body of scFv was isolated from E. coli lysate and solubilized in 6 M guanidine-hydrochloride without reducing agents, followed by refolding through slow dilution into refolding buffer. After complete removal of the remaining denaturant by dialysis, the soluble scFv was purified through an apo B-100-coupled affinity column, and an active fraction, which had an antigen-binding activity comparable with that of native Fab, was easily obtained. The expression and in vitro refolding of scFv resulted in production of an active molecule in a yield of 15-20 mg per 1-liter flask cultivation.
Lee
Myung-Hoon
MH
Genetic Engineering Department, Kyungpook National University, Daegu, Republic of Korea.
Park
Tae-In
TI
Park
Yong-Bok
YB
Kwak
Ju-Won
JW
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000097:blood / Q000737:chemistry*; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000276:immunology; D010957:Plasmids / Q000378:metabolism; D017510:Protein Folding
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D007135:Immunoglobulin Variable Region