Downstream technique study of human tumor necrosis factor alpha and its mutant

The study of downstream techniques for recombinant human necrosis factor alpha (rhTNF alpha) and its mutant (Lys2)-rhTNF alpha led to the results of approximately 50 g wet recombinant E. coli per liter with high expression level (> 50%) harvested from autocontrolled fed-batch culture in 15 L fermentor (B. Braun). The rhTNF alpha and (Lys2)-rhTNF alpha expressed are totally soluble. Followed by the process of ultrasonication, ammonium sulfate precipitation, ion-exchange chromatography (DEAE-sepharose FF, CM-sepharose FF), and molecular sieve (Sephacryl S-200), a yield of approximately 1 g pure recombinant protein from 1 L broth is obtained. The purity is up to 98%. The specific activity of rhTNF alpha and (Lys2)-rhTNF alpha are approximately 1.5 x 10(8) IU/mg and approximately 6 x 10(8) IU/mg, respectively.