Crystal structure of a family GT4 glycosyltransferase from Bacillus anthracis ORF BA1558
Crystal structure of a family GT4 glycosyltransferase from Bacillus anthracis ORF BA1558
复制标题
DOI：
10.1002/prot.22171
复制
发表时间：
2008-11-15
影响因子：
2.9
通讯作者：
Martinez-Fleites, Carlos
中科院分区：
文献类型：
--
作者：
Ruane, Karen M.;Davies, Gideon J.;Martinez-Fleites, Carlos
METHODSThe gene encoding BaGT4 BA1558 was amplified by the polymerase chain reaction (PCR) from Bacillus anthracis genomic DNA using primers corresponding to the predicted 5′ and 3′ ends and that incorporate 5′ overhangs designed to insert the gene into the pETYSBLIC vector. 8 The recombinant plasmid pETYSBLIC-BaGT4 BA1558 was introduced into BL21 (RIPL) E. coli cells and cultured in 0.5 L of Overnight Express Autoinduction System 2 for Se-Met labeling media (Novagen) supplemented with 50 mg L− 1 kanamycin, 34 mg L− 1 chloramphenicol and 50 mg L− 1 streptomycin at 37 C for 8 h. Protein expression was induced overnight at 25 C. Cells were harvested and resuspended in 20 mM HEPES pH 7.2, 400 mM NaCl and lysed by sonication. Resulting supernatant was applied to a 5 mL HisTrap column (GE Healthcare) where the protein was eluted with an imidazole gradient. Pure protein was buffer exchanged into 20 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM DTT and concentrated to 16 mg/mL for crystallization. BaGT4 BA1558 was crystallized from 0.1 M HEPES pH 7.5, 0.2M Na 2 SO 4, 14% PEG 3350. Crystals were cryo-protected in the mother liquor solution with the addition of 25% glycerol and flash frozen prior to data collection.
