title: Gene cloning and expression of MAP30 inPichia pastoris.
abstract: MAP30, a single-stranded type-I ribosome inactivating protein found inMomordica charantia, shows anti-HIV and anti-tumour activity. It could significantly inhibit the HIV-1 and herpes simplex virus infection. In this study, we tried a safe and convenient expression system supplying MAP30 protein for medical practice. The gene encoding MAP30 was cloned into pMD18-T vector. The pMD18-MAP30 plasmid was transformed into competentEscherichia coliJM109 by a chemical method. TheMAP30 gene was obtained from the pMD18-MAP30 plasmid digested withNotI andSnaBI and theMAP30 gene was ligated into pGAPHα. Then, pGAPHα-MAP30 was transformed intoPichia pastorisGS115 by electroporation. GS115 transformants were analysed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and Western blot. SDS-PAGE revealed an extra band of approximately 32 kDa in the supernatant protein of the GS115 transformants and in their intracellular protein fraction. The result of Western-blot analysis showed that the supernatant and the cell pellet from GS115 with pGAPHα-MAP30 could specially bind to monoclonal antibodies against His in the 32 kDa site. These results demonstrated that the expression of MAP30 inP. pastoriswas successful; the process of the expression did not need methanol induction or introduction of an antibiotic-resistance gene. The study may provide a new way for MAP30 synthesis. Owing to its safety, this new approach is expected to be widely used in the medical field.
authors.affiliation: Heilongjiang Provincial Key Laboratory of Plant Biology, Life Science and Technology College, Harbin Normal University , Harbin , China.
authors.forename: Fang
authors.initials: F
authors.lastname: Wang
authors.affiliation: Heilongjiang Provincial Key Laboratory of Plant Biology, Life Science and Technology College, Harbin Normal University , Harbin , China.
authors.forename: Chun-Yu
authors.initials: CY
authors.lastname: Chi
authors.affiliation: Heilongjiang Provincial Key Laboratory of Plant Biology, Life Science and Technology College, Harbin Normal University , Harbin , China.
authors.forename: Li-Yuan
authors.initials: LY
authors.lastname: Wang
authors.affiliation: Heilongjiang Provincial Key Laboratory of Plant Biology, Life Science and Technology College, Harbin Normal University , Harbin , China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Qiao
authors.affiliation: Heilongjiang Provincial Key Laboratory of Plant Biology, Life Science and Technology College, Harbin Normal University , Harbin , China.
authors.forename: Xiao-Xia
authors.initials: XX
authors.lastname: Jin
authors.affiliation: Heilongjiang Provincial Key Laboratory of Plant Biology, Life Science and Technology College, Harbin Normal University , Harbin , China.
authors.forename: Guo-Hua
authors.initials: GH
authors.lastname: Ding
keywords: MAP30; Pichia pastoris; clone; expression; pGAPHα