Generation of truncated recombinant form of tumor necrosis factor receptor-1 to produce cancer vaccine

摘要

Purpose: To produce truncated recombinant form of tumor necrosis factor receptor 1 (TNFR1), cysteine-rich domain 2 (CRD2) and CRD3 regions of the receptor were generated using pET28a and E. coli/BL21. Methods: DNA coding sequence of CRD2 and CRD3 was cloned into pET28a vector and the corresponding protein was expressed under induction of isopropyl β-D-1-thiogalactopyranoside (IPTG) as 6×His tagged using E.coli BL21 (DE3) ex pression system. The protein was then purified by Ni-NTA affinity chromatography. The fragment insertion, ex pression of recombinant protein and the yield of ex pression were evaluated. Results: Protein ex pression was achieved by identifying a band with molecular weight of 1488.3 Da. The recombinant protein of CRD2 and CRD3 was most efficiently expressed in 0.5 mM IPTG and 3 h of incubation at 37 °C with high yield equal to 0.3 μg/μl. Also, the highest concentration of imidazole for purification of the recombinant protein was 250 mM. Conclusion: A truncated form of TNFR-1 has been successfully expressed in a bacterial ex pression system and purified on affinity column. The purified protein can be used in in vivo experiments to prepare specified agonist antibodies for TNFR-1.