Expression, Puriifcation And Crystallization Of GNMT And Gale Proteins Of Bacillus Anthracis

Anthrax is an acute zoonotic infectious disease, mortality is high. Bacillusanthracis has often been used as a chemical and biological weapon, which threatenhuman survival and life safety of people, so the research of bacillus anthracis is stillan important subject faced by the world today.Glycine methyltransferase (GNMT, EC2.1.1.20) is abundant in nature both inprokaryotic and eukaryotic organisms. It is a key enzyme in amino acid metabolicpathways, which catalyzes glycine methylation into creatine. It can regulates-adenosine homocysteine and creatine ratio in the organism, playing an importantrole in biological body; So far, crystal structures of glycine methyltransferase wereobtained only from humans and mice. Other species of GNMT structure had not beenlearned in detail. So the studying of the crystalline structure of the enzyme frombacillus anthracis can make us understand the enzyme’s function and mechanism ofaction more comprehensive and in-depth. This can provide us the basis for applicationin drug design et al.UDP-galactose4-epimerase (GalE, EC5.1.3.2) is another important enzymewith great function in the body and is very widespread in nature too. It is the hubbetween glucose and galactose transformation, which acts a vital role in themetabolism of galactose; Currently, UDP-galactose4-epimerase crystal structure hasobtained from Human, Escherichia coli, Brinell trypan species; this provides the keyto uncover its structure and function mechanism, but large differences exsits amongdifferent species about this enzyme. Bacillus anthracis, as a kind of highly pathogenicbacteria, was studid to know the characteristics of the enzyme. It can make us morein-depth understanding of its biological characteristics and provide direction for drugdesign and disease diagnosis.According to GNMT (Registration number is BAS3318) and GalE (Registrationnumber is BAS1093) DNA sequence of prokaryotes of bacillus anthracis fromKEGG (the Kyoto Encyclopedia of Genes and Genomes) Database, their sequencelength were1356and846bp, respectively, with designed two pairs of specific primers, we obtained the gene which were consistent with the expected results afterPCR, and then we used double digests by restriction enzymes and connected them tothe expression vector pET-21a and pET-15b, after plasmid pcr, double digests andsequencing identification, we screened out the positive expression plasmid andtransformed them into E. coli BL21(DE3), built GNMT gene and GalE generecombinant expression vector of engineering bacteria successfully. After induced byIPTG, the target genes were expression in vitro. The recombinant proteins werepurified by Ni-affinity chromatography, Mono Q anion exchange columnchromatography and Sephacryl200(Sephacryl75) molecular sieve gelchromatography, and then we got enough fusion protein for the crystal research.Finally the protein crystallization experiment was carried on, the optimization crystalcondition was screened and single crystal was obtained, which provided reference forobtaining of GNMT, GalE high-resolution diffraction of single crystal, and laid afoundation for crystal structure analysis.