1. Expression and functional reconstitution of a recombinant antibody (Fab') specific for human apolipoprotein B-100
- Author
-
Myung Hoon Lee and Ju Won Kwak
- Subjects
Protein Folding ,Lysis ,Apolipoprotein B ,medicine.drug_class ,Bioengineering ,Antigen-Antibody Complex ,medicine.disease_cause ,Monoclonal antibody ,Applied Microbiology and Biotechnology ,Inclusion bodies ,law.invention ,Antibodies, Monoclonal, Murine-Derived ,Immunoglobulin Fab Fragments ,Affinity chromatography ,law ,medicine ,Escherichia coli ,Cloning, Molecular ,Cells, Cultured ,Apolipoproteins B ,Chromatography ,biology ,Chemistry ,Antibodies, Monoclonal ,General Medicine ,Gene Expression Regulation, Bacterial ,Molecular biology ,Recombinant Proteins ,Apolipoprotein B-100 ,Recombinant DNA ,biology.protein ,Immunoglobulin Light Chains ,Immunoglobulin Heavy Chains ,Biotechnology - Abstract
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.
- Published
- 2003