Induced Expression Of HIFNβ-HSA In Pichia Pastoris And Identification Of Its Degadation Protease

The yeast Pichia pastoris is a useful eucaryon expressing system which has developed in 10 years. This system has several good quality , for example, high amouts expression, clutivating low, product can secrete outside, proper glycosylation. At present, already have had more than 500 kind external protein expressed in Pichia pastoris. Proteolytic degradation has been a perpetual problem when yeasts are employed to express recombinant proteins. Alos this problem influent the accumulation of protein.On the basis of previously work, laboratory constructed the gene of Human IFNβ-HSA fusion protein, hIFNβ-HSA in Pichia pastoris KM71 by technique of gene engineering, which express the 90 kDa protein. And tthrough he effects of fermentative process parameter on cell growth and hIFNβ-HSA expression in 5 L fermenter were studied. On this basis , this research is about the problem of the Proteolytic degradation during the express process, the main results as follows:1. By the experiment of mono-factor, the main results as follows: under the pH5.0～pH6.0 the exprrssion of the protein is the highest, and 6% peptone was added to culture medium is the best, at the same time, Additions of Glu, Ala, Arg into the medium, 0.1% concentration has the optimization expression. According of the principle of Box-Benhnken, with the method of response surface of the three factors and three levels, the best addition program is pH5.5,Glu 0.18%,EDTA 12 mmol/L in the medium, obtained the amount of protein IFNβ-HSA is 24.87 mg/L, comparing with the blank referenc group, increasing 61%2.. The analysis of SDS-PAGE and Western-blot showed that 90kDa fusion protein and 65kDa degradation band appeared in the fermentation. By the gelatin-SDS-PAGE, three prteases inside and five proteases outside cell, and which degrdated the pritein. With the method of HPLC indicated that the degradation appeared in the HSA region of recombinant human IFNβ-HSA fusion protein. Applied by SDS-PAGE activity electrophoresis the protease which caused the degradation of fusion protein IFNβ-HSA was proteinase B...