Cloning And Expression Of A Serine Protease From Mycoparasite Fungus Olpitrichum Tenellum

Cell to cell recognition and signal transduction is an important area of life sciences research.The parasite relationship between mycoparasites and their host fungi exists in nature universally,makes it one of the best models to research the mechanism of interaction and signal transduction between two organisms. Olpitrichum tenellum is a biotrophic contact mycoparasite fungus.In last several years,our lab has establised a molecular interaction reaserch platform with it as a foundment.Cell wall is the first screen in cell to cell interacton.In recent years,reaserchers found the cell wall proteins play important roles in interaction of organisms,so the research of fungi cell wall proteins becames a hot spot and more and more attention was paid to the proteases of mycoparasitic fungi.In conclusion,cloning and expression of protease genes of mycoparasite fungi has a great significance.The serine protease is a group of proteolytic enzymes that are important for their potential applications. The members in the serine protease super family serve a wide range of physiological functions, especially in the pathogenesis of many diseases and intracellular signal transduction. Degenerate primers based on the conserved domain of other reported proteases were designed, and a cDNA fragment encoding the protease gene was obtained through RT-PCR. The RACE was used to generate the full-length cDNA clone. The full length of mppro cDNA gene is 2024bp, which contained an ORF of 1554bp encoding 517amino acids. The cDNA sequence of gene mppro has been registered in Genbank with accession number EU368754.We have analysed the cDNA sequence and amino acid sequnce of mppro with bioinformation technique.Through analysis,we found that mppro is a secretory protein,belongs to the serine protease, peptidase S8,subtilisin,has typical stucture of this family-a signal peptide,the propeptide,the catalytic domain,the P/middle or homoB domain and the C-terminus.We found that it shares high homology with Trichoderma ,which contains a lot of mycoparasitic fungi and maybe that is because they are all mycoparasitic fungi.The mppro gene and expression vector pET-22b(+) were digested with EcoRâ… a nd Notâ… , and ligation was carried out in vitro. The recombinant plasmid pET/mppro was generated and transformed into E. coli BL21. The recombinant protein was produced in large amount after IPTG induction, approximately 31kDa protein was detected by SDS-PAGE. No interest protein was produced by inducing with IPTG after the pET-22b(+) was transformed into BL21.But the expressed protein has no protease activity,maybe it is because the recombinant protein was presented in a fusion form.The mppro gene and expression vector pPIC9K were digested with EcoRâ… and Notâ… , and ligation was carried out in vitro. The recombinant expression plasmid pPIC9K/mppro was constructed and sequenced to confirm the correct reading frame. The constructed plasmid pPIC9K/mppro was linearized with a restriction enzyme XbaI (insertion at HIS4),transformed into Pichia pastoris GS115 competent cell by electroporation methods and screened for His+Mut+ transformants on MD and MM plates. The parent vector was linearized with the same restriction enzyme and transformed into GS115 as a control. P. pastoris integrants,selected by PCR analysis, are induced by methanol.After 5 days methanol inducing, the protease activity of P. pastoris integrants increased to 153U/mL.Just as the result of prediction by bioinformation technique,the protein's MW is 39kDa, determined by SDS-PAGE. The genetic stability of P. pastoris recombinant was tested.The recombinant protein is purified and characterized.