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.
