Heterologous Expression Of Human Glucagon Like Peptide-1 In Yeast System

Saccharomyces cerevisiae was used as a model organism by genetic engineering method of ribosomal protein synthesis initiation and depolymerization of artificial regulation to study host several target genes up-regulated expression mediated ribosome recycling acceleration of heterologous protein expression efficiency in this paper.Therefore,the ultimate goal of this study is to accelerate the ribosome cycle to enhance the expression efficiency of eukaryotic protein.In this paper,the effect of Tif3 gene up regulation on the expression efficiency of red fluorescent protein was verified by the effect of the expression of the key genes in the ribosomal protein synthesis.Firstly,the gene Glp-1-rfp;promoter CUP1 and termination of the CYC1 gene fusion,gene Qfh were synthesized.The plasmid YEplac112 and pMV-QFH were digested by restriction enzyme EcoRI and Hind?I.The enzyme digestion products was ligase by T4 ligase enzyme.The plasmid was named as YEplac112-P_CUP1-T_CYC1.The plasmid YEplac112-P_CUP1-T_CYC1 and pMV-RFP were digested by restriction enzyme PstI and BamHI.The enzyme digestion products was ligase by T4 ligase enzyme.The plasmid was named as Saccharomyces cerevisiae expression vector YEplac112-P_CUP1-GLP-1-RFP-T_CYC1.The expression of Glp-1-rfp gene was induced by copper ion in the transformed Saccharomyces cerevisiae W303 strain.Under the excitation of green light,it was observed that the cells of Saccharomyces cerevisiae could emit red fluorescence,which proved that Glp-1-rfp gene was successfully expressed in W303,which could be used as a reporter gene for subsequent experiments.Cloning of gene Tif3,promoter of Gal gene and terminator of Cyc1 gene was cloned by PCR.The plasmid YCplac33 and gene Gal were digested by restriction enzyme Hind?I and Xba I.The enzyme digestion products was ligase by T4 ligase enzyme.The plasmid was named as YCplac33-P_GAL;the plasmid YCplac33-P_GAL and gene Tif3 were digested by restriction enzyme BamHI and Pst I,The enzyme digestion products was ligase by T4 ligase enzyme.The plasmid was named as YCplac33-P_GAL –TIF3.The plasmid YCplac33-P_GAL-TIF3 and gene Cyc1 were digested by restriction enzyme SmaI and EcoRI.The enzyme digestion products was ligase by T4 ligase enzyme.The plasmid was named as Saccharomyces cerevisiae expression vector YCplac33-P_GAL-TIF3-T_CYC1.The expression of Rfp gene and Tif3 gene was induced by semi lactose and copper ions.In by the same time,under the excitation of green light observation of recombinant Saccharomyces cerevisiae cells,can only transform the Glp-1-rfp gene of the Saccharomyces cerevisiae expression induced by cells from the obvious red fluorescence.It is proved that the successful expression of Glp-1-rfp gene in Saccharomyces cerevisiae W303;at the same time the transformation of Saccharomyces cerevisiae gene Rfp and gene Tif3 after induced expression of cells from the higher fluorescence intensity of red fluorescence.It is proved that the gene Tif3 and gene Glp-1-rfp also in Saccharomyces cerevisiae W303 was expressed,and the upregulation of gene Tif3 expression increased the efficiency of exogenous protein expression of gene Glp-1-rfp.