Expression And Bioactivity Assay Of Alpha-momorcharin Gene In Pichia Pastoris

α-MMC (alphα-momorcharin, α-MMC) is a type I ribosome-inactivating protein in the seeds of the bitter gourd (Momordica charantia). It has a number of biological activities including induction of abortion, inhibition of tumor growth, antivirus and antibacterial. α-MMC is a type of glycoproteins, it was initially synthesized as a 286 amino aced peptide, with a 23-amino acid signal peptide and a 263 amino acid mature peptide, containing a sugar chain which is bound to Asn-227, a side chain which is located at Trp-70 acting as a switch contributing to the valve, of the active center Glu-160. α-MMC possesses several types of enzyme activity, such as N-glycosidase, RNase and deoxyribonuclease (DNase) activities. All its biological activities may be related to the ribosome-inhibiting activity of the protein. In this study, we product recombine a-MMC to set a fundation for further research and application of α-MMC.To obtain the encoding gene of α-MMC is the bases of this whole study. Using the isolated total RNA from the flash leaves of Momordica charantia as a template, the cDNA encoding α-MMC was amplified by reverse transcription-polymerase chain reaction (RT-PCR) method. The PCR product was cloned into pGEM-T Easy Vector and sequenced, and the α-MMC cDNA was obtained successfully, named as α-MMCC. A recombinant expression plasmid pPICZaB/a-MMCC was constructed and transformed into yeast host strain GS115, a kind of Pichia Pastoris. Recombinant and Mut phenotype was confirmed by PCR methods. And then quested for the methanol inducement conditions by the orthogonal experiment. The recombine protein was confirmed by SDS-PAGE and Western blot anlysis. At the optimized inducement condition (BMGY pH6.0, BMMY pH6.5 1.0% methanol, inducement period 72h), the espression levels were estimated to be 15% of total proteins in supernatant.The recombine expression product was purified by FF affinity chromatography, after that its purity came up to 90%. The results of MTT assay proved that recombinant a-MMC possessed the natural biological activity, induced Hela apoptosis in a time-dependent manner. And the molecular mechanism with which α-MMC induces cell apoptosis needs to be further reseached.