Generation of truncated recombinant form of tumor necrosis factor receptor-1 to produce cancer vaccine
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) expression system. The protein was then purified by Ni-NTA affinity chromatography. The fragment insertion, expression of recombinant protein and the yield of expression were evaluated.
Results: Protein expression 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 expression system and purified on affinity column. The purified protein can be used in in vivo experiments to prepare specified agonist antibodies for TNFR-1.
Keywords: Tumor necrosis factor receptor 1 (TNFR-1), Cysteine rich domain 2 (CRD2), Cysteine rich domain (CRD3), Apoptosis, Cancer vaccine, Antibodies, Recombinant protein, pET28a