Heterologous Expression Of MrpF And Its Structural And Functional Research

Mrp(multiple resistance and pH-related antiporter) are proposed to be uniquely complex multi-subunit monovalent cation/proton antiporters, encode seven or six hydrophobic proteins that are likely to be coordinately expressed as an operon. This issue focus on the membrane protein MrpF, one member of the complex, has three transmembrane segments with molecular weight of 11.2 KDa. It plays an essential role in cytoplasmic pH and osmotic pressure homeostasis and bile salt resistance.Based on the MBP tag we had successfully expressed fusion protein MBP-MrpF, we studied and analyzed the feasibility of the intein, a novel self-cleavaged tag, in the membrane protein. Intein is a popular polypeptide; it can achieve self-cleaving during the process of protein purification. This property can simplify the traditional ways to purify protein. Meanwhile membrane protein is always a research hotspot. Through this research, we expect to set up a simple and convenient pipeline, so as to optimize the purification of membrane protein. By constructing and expressing fusion protein MBP-Intein-MrpF, we change the condition of pH or temperature to verify the self-cleavage ability of intein in MrpF.Also, we have achieved heterogeneous expression of MrpF in E.coli. By optimizing various expression conditions, such as expression vector, expression strain, expression temperature and induced time, cell density, carbon source and concentration, nutrition additive and so on, we have obtained a set of expression conditions to achieve overexpression of MrpF, that is, cloning the mrpf into the expression vector pET28 OH and transformed into the expression train C43 to express the fusion protein 6His-MrpF. In the M9 minimum media contain 4g/L glucose and trace mental, we cultivated the bacteria until the OD600?0.8. By adding the IPTG to the concentration of 1 mM, the fusion protein were induced at 25 for ?16 h. Eventually we achieved heterologous excess expression of MrpF, and the production is 8-9 mg/L. On this basis, CD and FTIR have used to explore the secondary structure of MrpF in detergent solution. And we got mainly ?-helix which is consistent with the the theoretical structure. On the other hand,we also use FTIR,liquid NMR and ITC to verify the interaction between MrpF and cholate salt?At last, this tissue screen different kinds of detergent so to find the most suitable one to stable and dissolve the MrpF.