Expression, purification and investigation of sap۲ Candida albican’s activity in pichia pasturis yeast system

Objective: The secreted aspartic proteinases (Sap۲) of Candida albicans has prominent role on Candida adherence, invasion, and pathogenicity. The aim of this study was cloning, expression and characterizing of Sap۲ enzyme. Also in this study for the first time, the expression system P. pasturis was used for expressing the recombinant protein. Materials and Methods: C. albicans Sap۲ gene was amplified by PCR with sticky ends, EcoR۱ and SacII, and it was subcloned into the T/A vector. The sequencing of this gene was done with universal primers and then the Sap۲ gene was cloned into pGAPZαA expression vector. The construct was transformed into P. pasturis yeast; the Sap۲ gene integration into the yeast genome was accomplished by the homologous recombination. The expressed protein was confirmed by western blotting using monoclonal antibody against Sap۲ protein. Finally, the recombinant protein was purified by Ni-NTA chromatography column, and the activity of the enzyme was confirmed. Results: In this study, we successfully amplified C.albicans Sap۲ gene and subsequently integrated into the yeast pichia pasturis genome by homologous recombination. Moreover, we were able to identify a yeast clone secreting the recombinant protein. The optimum over expression of sap۲ protein was obtained after ۹۶ h, at ۳۰ C. Conclusion: Expression of Sap۲ gene in P. pasturis, in comparison to bacterial expression system, leads to a high-level expression, and also need for post translation modifications, that might be required for the activity of enzyme, is obviated in the yeast system. Based on our results, the purified acid aspartyl proteinase purified from P. pasturis was capable of degrading BSA as a substrate in-vitro. The recombinant Sap۲ protein had maximum activity in an acidic pH.

کلیدواژه ها:

C. albicans ، Sap۲ cloning ، P. pasturis ، کاندیدا آلبیکنس ، کلونینگ Sap۲ ، پیکیا پاستوریس