The Research Of EGFP Transmembrane Transport By Tat Pathway Of Staphylococcus Carnosus

Enhanced green fluorescent protein(EGFP)can be expressed in a variety of prokaryotic and eukaryotic cells,its expression is not restricted by biological type,genotype,or tissue type.EGFP is widely used in various fields of life sciences because it has many characteristics,such as easy to detect,high sensitivity,stable fluorescence property,non-toxic to cells,and direct application for live cell assays.Staphylococcus carnosus is a good host bacterium because it does not produce any toxin,hemolysin,coagulase and has low extracellular proteolytic activity.The signal peptide is needed in transportation of the protein which is synthesized in the cytoplasm.Currently in genetic engineering,the Sec secretion pathway is commonly used for exogenous protein transportation,but it can only transport unfolded or partially folded protein,while Tat translocation system can secrete properly folded protein.Thus Tat pathway can secrete some proteins which can not be secreted by Sec pathway.A shuttle vector has two different replicators,which can replicate in two or more different hosts.In bacteria,the shuttle vector is generally used for gene amplification.In this research,the egfp gene fragment containing 20 kinds of a 5-same amino acid linker at N-terminal(5x-egfp fusion gene)was cloned into E.coli-Staphylococcus shuttle vector pBT2-ET-EGFP,then the recombinant plasmid pBT2-ET-5X-EGFP was electrotransformed into S.carnosus TM300 host to study the effects of Tat pathway on the expression and secretion of exogenous protein.After the fermentation culture of S.carnosus TM300/pBT2-ET-EGFP and S.carnosus TM300/pBT2-ET-5X-EGFP,the target protein EGFP was observed active in vivo by fluorescence microscopy and the fluorescent cells can be also detected by flow cytometry,indicating that EGFP was successfully expressed.SDS-PAGE and Western blot were used to detect the EGFP in the cell wall and the supernatant.The result revealed that EGFP from both S.carnosus TM300/pBT2-ET-EGFP and S.carnosus TM300/pBT2-ET-5X-EGFP was successfully secreted to the cell wall in the both forms of monomer and dimer.Meanwhile,the EGFP from recombinant S.carnosus TM300/pBT2-ET-5X-EGFP was released into the supernatant only in the dimer form.Fluorescence spectrophotometer assays showed that the EGFP was in active form in the cell wall,but not detectable in supernatant.Furhtermore,the EGFP concentration in the supernatant was determined at about 100?150 ng/L using ELISA(Enzyme-Linked Immunosorbent Assay)assay kit.