Tan_2010_Zhongguo.Wei.Sheng.Tai.Xue.Za.Zhi_22_1061

Objective: To construct an Pichia pastoris expressing vector pPIC9-ZEN-jjm and screen the strains which could express high-level active proteins. Method: ZEN-jjm gene was spliced into pPIC9 after digested with EcoR I and Not I, then transformed into Pichia pastoris GS115 by electroporation. The recombinant yeast strains were screened with RDB medium and methanol inducible expression. The ZEN degradation capabilities of expressed supernatant was verified by HPLC test. Result: DNA sequencing demonstrated that ZEN-jjm was inserted into pPIC9. SDS-PAGE demonstrated that one yeast strain with high-level expression was obtained, and the molecular weight of the expressed protein was about 29 kDa. The HPLC result showed that the expressed protein could effectively degrade ZEN. Conclusion: ZEN-degrading enzyme is highly expressed in Pichia pastoris.