Study On The Construction Of Photosynthetic Bacterial Membrane Protein Expression Vector

Protein is a major component of the body's cellular structure,signal transduction and molecular transport,and is one of the important research contents in the field of modern life sciences.Escherichia coli and yeast ar e the most commonly used and most classical expression systems.When expressing foreign proteins,especially when expressing membrane proteins,proteins often fail to fold correctly,easily form inclusion bodies,and lack biological activity.Moreover,when most of the expression systems currently used express proteins,it is often impossible to perform real-time quantitative detection of recombinant proteins.The oldest bacteria of photosynthetic bacteria,safe and non-toxic,can be used as a new type of expression system.Rhodospirillum(R.)rubrum is a purple non-sulfided facultative bacterium that synthesizes the cytoplasmic intima(ICM)with photosynthetic apparatus in the absence of oxygen.It can bring proteins to the cell membrane to form membrane proteins.The problem of membrane protein expression is difficult.Thermochromatium(Tch.)tepidum is a high-temperature purple-sulfur bacteria with a unique light-harvesting pigment protein LHI with an absorption peak at 915 nm,which can be used as a reporter gene.In this study,R.rubrumH2 was used as a membrane protein expression host,and the LHI encoded by Tch.tepidum pufBA was used as a reporter gene.This expression system was verified by detecting DAA.Firstly,pufBA was recombined with pPUCTerm vector which can be expressed in pHotosynthetic bacteria by gene recombination technology,and transformed into R.rubrumH2 by E.coli WM3064.By spectroscopic and electron microscopy,it was found that pufBA was normally expressed in R.rubrumH2 and existed in the form of membrane protein,and an absorption peak appeared at 915 nm.The DAA is then recombined with the pPUCTerm vector containing the reporter gene pufBA and transformed into R.rubrumH2.It was found that pufBA and DAA were successfully expressed to form a fusion protein,the position of the absorption peak remained at 915 nm,the absorption peak is positively correlated with the protein expression and the expression product was active.In this study,the R.rubrumH2 membrane protein expression system containing the reporter gene pufBA was successfully constructed,which provided a new idea for real-time detection of protein,and has good value in protein expression and real-time detection.