Construction Of Recombinant SCYL1-BP1 Gene Engineering Strain And Its Expression

A novel human gene SCYL1-BP1,which can bind to NTKL,was cloned from placenta cDNA library in our laboratory by yeast two hybrid system. Our group’s early study found: This protein is mainly distributed in the intranuclear, a small amount of the distribution in the perikaryon and cytoplasm. The colony formation experiment, the result of cell growth curve and in vivo tumorigenicity assay all showed over-expression of SCYL1-BP1suppress the growth of liver cancer cell and the tumor formation. The excessive amounts of SCYL1-BP1proteins could decrease cell proliferation by interfering the G2/M progression. Increases in SCYL1-BP1protein levels in cells cause parallel changes in the amount of p53protein, which coming from the inhibition of MDM2-mediated p53ubiquitination. Ch1P-on-chip experimental results showed that the expression of NFκB would rise when the gene was inhibited by RNAi. Reports showed that the excessive activation of NFκB will result in tumor. The results suggested that SCYL1-BP1seemed to play an important role in the cell cycle and tumor suppression.In order to obtain a sufficient amount of purified SCYL1-BP1for the new drug safety testing and a series of experiment of pharmacology, we plan to use genetic engineering techniques to expressed the protein in a large amount.This thesis is composed of two separate parts:the expression of human protein SCYL1-BP1in E. coli by the way of genetic engineering, and the purification of this protein; the study on the separation and purification of recombinant protein SCYL1-BP1in Pichia pastoris expression system.In part I, the SCYL1-BP1gene was inserted into the high expression vector pET-28b-sumo to construct recombinant prokaryotic expression plasmid pET-28b-sumo-SCYL1BP1, which was transformed into E. coli to be expressed after IPTG induction. In order to get the high expression, we detected the consideration of the expression, the choice of the host E.coli, the concentration of IPTG, the time of culture after inducation. Finally we found that the results of prokaryotic expression is not good,maybe because of the protein’s cell toxicity.In part Ⅱ,according to the codon preference of P. pastoris and the amino acid sequence of SCYL1-BP1, we get the full length of the gene through the technology of overlapping PCR. The full length gene cloned in the expression vector pHBM905A,was transfected into P. pastoris strain GS115as pHBM905A-SCYL1BP1.After48hours culture in BMGY, then get over the culture, transferred into the BMMY for the inducation expression. Induce with1%methanol every24hours. The optimal harvesting time of cells was determined as48-60h after induction. SDS-PAGE showed that the protein was successfully expressed as a secretory protein in P. pastoris and showed as between50kDa and60kDa bands.