Overexpression, effective renaturation, and bioactivity of novel single-chain antibodies against TNF-alpha.
Neutralization of tumor necrosis factor-alpha (TNF-alpha) has become an effective therapeutic strategy for TNF-related autoimmune diseases. Due to the limitations of the large molecular inhibitors in the therapy, development of novel TNF-alpha inhibitors is very attractive and useful. In this study, based on the previously designed domain antibody, two novel human anti-TNF single-chain antibodies were constructed using modular consensus frameworks of human antibody as scaffold to display the antagonistic peptides. A variety of expression plasmids were used to determine the optimal expression system. The single-chain antibodies were always overexpressed in E.coli BL21(DE3) host as inclusion bodies. Under the optimized refolding conditions, the inclusion bodies were renatured successfully and the refolded single-chain antibodies could bind with TNF-alpha and block TNF-induced cytotoxicity on L929 cells. The bioactivity of the single-chain antibodies was significantly increased over the domain antibody.
Geng
Shusheng
S
Hebei University, Baoding, PR China.
Chang
Hong
H
Qin
Weisong
W
Li
Yan
Y
Feng
Jiannan
J
Shen
Beifen
B
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001327:Autoimmune Diseases / Q000628:therapy*; D015153:Blotting, Western; D002460:Cell Line; D003602:Cytotoxicity, Immunologic / Q000187:drug effects*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D008969:Molecular Sequence Data; D020673:Protein Renaturation / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000276:immunology / Q000494:pharmacology
D000906:Antibodies; D014409:Tumor Necrosis Factor-alpha