The Cloning, Expression, Purification And Crystallization Of Glycosyltransferase MoeGT1

Glycosyltransferases catalyze glycosidic bond formation using sugar donorscontaining a nucleoside phosphate or a lipid phosphate leaving group. A family GT1enzyme MoeGT1involved in the biosynthesis of the antibiotic moenomycin has notbeen subjected to structural. Moenomycin is regarded as a antibiotic targetingtransglycosylase and destroying the formation of cell wall. Moenomycin has long beenwidely used in animal feed, healing stomach ulcers and treating the infection of theGram-negative bacteria. The emergence of resistance to existing antibiotics representsa significant threat to public health. This research gives us a chance to generate newvarities of moenomycin analogues. We aim at obtaining structure by X rey diffractionand finding the glycosylation site. The structure will help us develop new drugs.MoeGT1(Streptomyces ghanaensis ATCC14672) was obtained by artificial genesynthesis introducing NcoI and XhoI restriction sites. The artificial gene synthesisproduct was digested with NcoI and XhoI, and ligated using T4DNA ligase (NEB) intolinearized vector pET-32a(+) to give N-terminal2×Strep-tagged pET-32a(+)-MoeGT1-5AG. In order to improve protein solubility and get properly folded and biologicallyactive protein, I made use of BL21trxB (DE3) as host strains. The strain facilitatesdisulfide bond formation in the cytoplasm of bacterium when expressing protein haspotential disulfides. A purification strategy was designed by incorporating a His-tag onMoeGT1for affinity purification and a TEV protease cleavage site to generate the finalproduct. In order to increase the purity of MoeGT1, we need Ni2nd affinitychromatography and DEAE ion exchange chromatography in the following steps. Andwe finally obtain2.2mg target protein with purity above95%.Because of the low efficiency in TEV protease digesting, I speculated that muchlonger linker between TEV protease cleavage site and MoeGT1could completelyexpose cleavage site and increase the efficiency of digestion. After adding6×His-tagand5×linker amino acid (AGAGA) between TEV protease cleavage site and MoeGT1, the length of digestion was reduced from48hours at4oC to9hours at4oC. pET-32a(+)-MoeGT1-6H-10AG was successfully expressed by lowering induction temperature andusing auto-inducing ZYM-5052medium. Cell mass and target protein yield are oftenincreased several-fold as compared with conventional protocols using induction withIPTG. Target protein MoeGT1was purified from the supernatant by three steps: Ni1staffinity chromatography, Ni2nd affinity chromatography and DEAE ion exchangechromatography. We finally get18mg target protein with purity above95%.The MoeGT1protein was screened by using the screen kits from Hampton Research,Emerald BioSystems and Molecular Dimensions. Preliminary screening adoptedsitting-drop vapor diffusion method with48wells plate and optimized screeningadopted sitting-drop and hanging-drop vapor diffusion method with24wells plate.Finally, we find MoeGT1: FPG aggregation without closely packing. Though we didnot get the structure, there are lots of methods to improve the situation. We will spareno effort to try new ways; for instance, cocrystallization with crown compounds,regulating the temperature and changing the protein sample into Sumo-MoeGT1.