The Studies Of The Expression Of Human Cu,Zn-SOD In Pichia Pastoris

Superoxide dismutase plays a very important role in the body, it is a metal enzyme which is widely spread in the various organizations organisms. Pichia pastoris is a nutrition-type yeast.In this type, to study the expression of Human Cu,Zn-SOD in Pichia pastoris, explore the optimal conditions for expressing Human Cu,Zn-SOD and determine the stability of expressed SOD to chemical reagents, temperature and ph.Synthesis Human Cu,Zn-SOD gene by chemistry method, the gene was inserted into the Pichia pastoris expression vector pPIC9K which contained A0X1 promoter and secreting signal petides, the recombinant expression plasmid pPIC9K/SOD was constructed and transformed into GS115. The identified recombinant GS115/SOD was fermented in flasks. Furthermore, the expression conditions were explored for the time of expression, pH and final concentration of methanol, the stability of expressed SOD to chemical reagents, temperature and pH have been tested. SDS-PAGE showed that the product with related molecular weight of 18 KD was secreted into the supernatant in the culture. Western blot assay further proved that Human Cu,Zn-SOD exists in the expressed proteins of GS115/SOD. The highest quantity of SOD in the supernatant achieved at 91mg/L. The highest activity of supernatant reached at 334U/ml. The optimal fermentation conditions are at pH6.0 and 1.5% (v/v) methanol concentration and cultivation for 96 hours. The expression of secreted Human Cu,Zn-SOD with biological activity was successfully achieved in Pichia pastoris expression system, the optimal fermentation conditions and the stability of expressed SOD to chemical reagents, temperature and pH have been determined.