Zhang M.-Y.,Wuxi MTLH Biotechnology Co. |
Huang L.,Wuxi MTLH Biotechnology Co. |
Li Z.-F.,Wuxi MTLH Biotechnology Co. |
Guo M.-J.,Wuxi MTLH Biotechnology Co. |
Zhou H.,Wuxi MTLH Biotechnology Co.
Chinese Journal of Biologicals | Year: 2013
Objective: To express small TNFα antagonist (STNFαA), with a low relative molecular mass, binding to tumor necrosis factor receptor 1 (TNFR1), and determine the biological activity of purified protein. Methods: The STNFα A amino acid sequence with high binding ability to TNFR1 was designed via computational optimization, based on which target gene was designed and synthesized according to the codon bias of E. coli, than inserted into plasmid pET-28a(+). The constructed recombinant plasmid pET28-STNFαA was transformed to competent E. coli BL21 (DE3) and for expression under induction of IPTG. The expressed product was identified by SDS-PAGE, purified by Ni Sepharose 6 Fast Flow chromatography, then analyzed by SDS-PAGE and HPLC, and determined for biological activity by MTT method. Results: Restriction analysis showed that recombinant plasmid pET28-STNFαA was constructed correctly. The expressed STNFαA, with a relative molecular mass of about 17 000, contained about 25% of total somatic protein, mainly existed in a form of inclusion body, reached a purity of more than 95%, and showed dose-dependent inhibitory effect on TNFα. Conclusion: STNFαA was expressed successfully, which inhibited the cytotoxic biological activity mediated by TNFα. It laid a foundation of development of novel TNF antagonists. Source