Optimization Of Tat Peptide And Research On Ts Function Of Transmembrane Transport In Staphylococcus Carnosus

Signal peptide is needed for secretion of protein after synthesized in cell cytoplasm.Currently the most commonly used Sec Translocation Pathway signal peptides only deliver unfolded or partially folded proteins to the extracellular environment while the Twin-Arginine Translocation(Tat)Pathway signal peptides are able to transport properly folded proteins,,which has made it a focus of protein transportation research wordwidely.Due to its low extracellular proteolytic activity,the generally regarded as safe stain Staphylococcus carnosus is a good host bacterium and can be served as a perfect tool for rescearch on transmembrane transport of exogenous protein via Tat Pathway and relative metabolic pathway in S.carnosus TM300.Enhanced green fluorescent protein(EGFP),secreted alkaline-phosphatase(SEAP),and firefly luciferase(FLUC)are three widespread reporter proteins with different advantages.Establishment of double reporter protein system is a clever combination of two features.This research used S.carnosus/pBT2-ET-5R-EGFP as original strain,An optimization of Tat peptide was firstly applied via PCR.By analyzation of SDS-PAGE,fluorescence microscope observation and flow cytometry(FACS),EGFP was expressed in an active form of monimer in the cells of S.carnosus,Most of which was trapped in the cell wall while only a little was secreted to the extracellular environment in a form of dipolymer without activity.The C-terminal modification of Tat by 4 amino acid prolongation and 3 same amino acid linker increased translocation efficiency by 17%,respectively,meanwhile three refolding relative mutants not only increased the transfer efficiency by 38%,145%and 151%,but also raised EGFP activityFurther,firefly luciferase which has strong enzymatic amplification effect and a EGFP-SEAP double report system with both benefits of easy to observe and fit for quantification was applied to investigate the transmembrane transport function of Tat Pathway.In the recombinant strains without Tat,SEAP was only detected in the protoplast,which means SEAP synthesized in the cell cannot be transported outside cell membrame,while both high activities of SEAP and FLUC were detected in the fermentation broth of recombinant strains with Tat,indicating that SEAP,EGFP-SEAP and FLUC could be delivered out of cell wall via Tat Pathway in S.carnosus.The research established a plasmid model for Tat secretion pathway in S.carnosus with EGFP,SEAP and FLUC as report proteins The research revealed that transmembrane transport of exogenous protein via Tat Pathway in S.carnosus TM300 acts an important role,and laid the foundation to further reveal the mechanism of Tat-protein transportation in other microorganisms.