Fusion Expression and Biological Activity Analysis of ESAT-6 Protein in Pichia pastoris

The object is to obtain the high-level expression of ESAT-6 protein secreted by Mycobacterium tuberculosis in Pichia pastoris. Human IFN α-2a and esat-6 genes were amplified by PCR. The DNA linker between the two genes encodes the polypeptide which is specifically recognized by enterokinase. The recombinant genes were inserted into Pichia pastoris expression vector pPIC9K containing AOX1 and α secreting signal peptides, the recombinant plasmid was constructed and transformed into Pichia pastoris SMD1168. The high expressed transformants obtained by G418 screening were fermented in flasks and induced by 1% methanol. After 4 days methanol induction, the recombinant protein was identified by SDS-PAGE, Western blot and biological activity of α-IFN. The SDS-PAGE and Western blot analysis showed that the molecular weight of expressed recombinant protein was about 30kD and the expressed protein could specifically react with the serum of tuberculosis patients. The expressed protein was also proved to react specifically with anti-αIFN antibody and have high biological activity of IFN. High-level expression of secreted IFN-α-2a-ESAT-6 fusion protein with good biological activity was successfully achieved in Pichia pastoris expression system, which could be applied to diagnose tuberculosis and develop TB new vaccine.