Prokaryotic expression of extracellular domain of B7.2 (CD86) and fermentation study on the engineered bacteria

Objective: To express the extracellular domain of human B7.2 (CD86), B7.2 (IgV + C), in prokaryotic expression system and to optimize the fermentation for engineered bacteria. Methods: B7.2 (IgV + C) was amplified by PCR from B7.2 whole cDNA, and then was cloned into an expression vector pGEX-4T-3 to form a recombinant pGEX-4T-3/hB7.2 (IgV + C). SDS-PAGE and Western blot were performed to identify the expression of the target protein. The optimal induction duration and the optimal inducer concentration were also selected and the inheritance stability of the recombinant plasmid was studied. Results: After being transformed into E. coli DH5α, recombinant pGEX-4T-3/hB7.2 (IgV + C) was induced by IPTG to express a 55 Kda fusion protein in E. coli at a level of about 30% of the total cellular protein. The fermentation study showed that the recombinant plasmid was inherited steadily in DH5 α, with a stable expression yield of targeted protein and without loss of recombinant plasmid, and the expression yield peak could be seen at 5 hours after induction and 4 mmol·L^-1 IPTG concentration. Conclusion: These findings presented the possibility of expressing the human B7.2 (IgV + C) molecule in prokaryotic expression system.