Cloning And Expression Of Sacchromycopsis Fibuligera β-Glucosidase Gene In Pichia Pastoris

To research expressing efficiently antitumor drugs, such as TNF, IFN and so on, in the future, Sacchromycopsis fibuligera (-glucosidase gene (BGL1) was cloned and was expressed in Pichia pastoris. Two pair of primers were designed bassed on the sequence of Sacchromycopsis fibuligera (-glucosidase gene (BGL1) and the sequence of multiple cloning site in pPIC9K which is a expression vector of Pichia pastoris. The PCR products of SHF and SHR were linked into pGEM-T vector and the plamid pSHLTV was developed. After the target gene was cut down by restriction endonuclease from pSHLTV, BGL1 was cloned downstream to the alpha-factor, and its open reading frame was in frame with the alpha-factor signal sequence in pPIC9K. The recombinant plasmid pSHL9K was transformed into Pichia pastoris GS115 by electroporation. The recombinant (-glucosidase is steadily secreted by Pichia pastoris strains. The recombinant strains contained the most multiple copy of pSHL9K were selected by culturing in media with G418. The optimum temperature and the optimum pH of the recombinant (-glucosidase are 50℃ and pH 5.4,respectively. While the recombinants were cultured in optimum expression condition, the amount of total secreted proteins is above 9g/L and the activity of (-glucosidase is 47 IU/mL in the culture medium. The relative molecular weight of the recombined (-glucosidase is about 113 kDa on 10% SDS-PAGE. can play important roles by means of participating antitumor processes, directly or in- directly, such as hydrolyze cellobiose, glucosylceramide, the sugar linamarin and so on. The lack of the (-Glucosidase would lead to accumulation of glucosylceramide and Gaucher disease. (-Glucosidase, also, can be used in the diagnosis and treatment of tumor. Pichia pastoris can efficiently express some cytokines, such as TNF, IFN, etc. This expression system was studied furthermore in the experiment and it will be a candidate system to expressing much more drugs of antitumor. It is the first time that the (-glucosidase gene of Sacchromycopsis fibuliger was expressed in Pichia pastoris up to now. Some stable recombinants which can produce (-glucosidase effiently are obtained and it is assured by some experiments.