The Expression And Purification Of Bacillus Subtilis Manganese Transporter Mnth

The manganese transporter MntH that exists widely on the membrane of variesof bacteria uptakes Mn2+into cytoplasm from the environment by the proton electro-chemical gradient as the driving force. Studies indicate that MntH also uptakes Fe2+,Zn2+, Co2+, Ni2+besides Mn2+. The MntH in bacteria is a homologue of the divalentcation transporter natural resistance-associated macrophage protein-1(NRAMP1)family in eukaryocyte. Ferrous ion and divalent manganese ion play a important roleboth in eukaryocyte and prokaryocyte, such as acting as a prosthetic group of enzymesparticipating in various of redox process, and affect the toxicity and pathogenicity ofthe bacteria,while in human they also play a critical role in infection and immunity.Currently, a complete refined structure of the NRAMP family dosen’t occur whateverin eukaryocyte or prokaryocyte, and the detailed mechanism how they transportdifferent divalent cation by proton electro-chemical gradient remains unknown. MntHis a low-expressed membrane protein transporter in bacteria. However, a quantity ofhighly purified proteins is required in obtaining crystals by X-ray crystallographymethods. Consequently, how to improve the expression quantity of the membraneprotein MntH remains a problem urgently needed to be addressed.The mntH gene was cloned from the gram-positive bacteria Bacillus subtilis andwas expressed in BL21(DE3) strain by pET15b plasmid. Using the Ni-NTA affinitychromatography, membrane protein MntH is purified and separated further viamolecular-exclusion chromatography by FPLC system. During this process, DDM,CHAPS, LDAO, Triton X-100, OG, CYMAL5and some other detergents are testedto stabilize MntH. In consideration of the low expression, MBP and SUMO taggedfusion proteins are tried to improve the yield of MntH in bacteria. The extractionefficiency of MBP/SUMO-MntH is detected by12detergents in a10-fold criticalmicell concentration(CMC).The TEV protease is used to digest the fusion protein fora higher purity, which is a necessary condition to crystallize. The purification processis improved by changing detergents from wash steps in order to make MntH stabledin a detergent with a better stability and monodispersion.In our syudy, it is found that detergents DM, DDM, LDAO, Triton X-100,CYMAL5, Fos12have a better extraction efficiency of the fusion protein and LDAO,DDAO, OG, Triton X-100have a trend to have a better monodispersion in molecular-exclusion chromatography.With these studies,we can purify a large number of highlypurified membrane protein MntH,and it can be monodispersed better than now withoptimization,which is beneficial to the next crystallization process.