Expression and functional reconstitution of a recombinant antibody (Fab') specific for human apolipoprotein B-100.
We have cloned and constructed plasmid vectors, pETB23H and pETB23L, for bacterial expression of heavy (H) and light (L) chain cDNAs of Fab' of mAbB23 a monoclonal antibody specific to human plasma apolipoprotein (apo) B-100. The H- and L-chains were expressed as insoluble inclusion bodies in the cytoplasm of Escherichia coli. The inclusion bodies of both chains were isolated from the cell lysate, solubilized in 6 M guanidium-HCl, and mixed in equal molar amounts. Refolding was performed in three stages of dialysis: first, dialysis against 3 M guanidium buffer, next, continuous decrement of guanidium in the dialysis buffer through slow addition of 1 M guanidium buffer, and finally, dialysis against a buffer without guanidium. After the refolding, active Fab' (rFab') was purified through an apo B-100-coupled affinity column. When compared by ELISA, the rFab' had a slightly decreased antigen-binding activity (about 0.7-fold) compared with native Fab. The refolding yield was maximum (75%) when performed at the protein concentrations not more than 0.4 mg ml(-1), whereas the yield decreased exponentially at higher concentrations. The maximum recovery was obtained at the refolding concentration of 1.8 mg ml(-1), where the yield was about 45%. Overall, 2.4-3.0 mg of active rFab' specific to apo B-100 was successfully obtained from 1 l cultivation of E. coli cells.
Lee
Myung-Hoon
MH
Cardiovascular Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, PO Box 115, Yuseong, Daejeon 305-600, South Korea.
Kwak
Ju-Won
JW
D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D058846:Antibodies, Monoclonal, Murine-Derived; D000936:Antigen-Antibody Complex; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000276:immunology; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D000936:Antigen-Antibody Complex; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins; C099704:heavy chain, monoclonal antibody B23; C099703:light chain, monoclonal antibody B23