腾冲嗜热厌氧菌新型变旋酶的表达、纯化及晶体分析

目的 纯化腾冲嗜热厌氧菌新型变旋酶TTE1925,用于晶体生长和三维结构分析.方法 将tte 1925基因克隆至原核表达载体pGEX-6P-1中,将菌落PCR和测序鉴定正确的重组质粒转化E. coli BL 21(DE 3)后获得表达菌株.该菌株经异丙基-β-D-硫代半乳糖苷诱导高效表达出带有谷胱甘肽S转移酶标签的可溶性融合蛋白,经过Glutathione Sepharose~(TM)4B亲和层析、Resource Q 6mL离子交换层析和10/300 superdex 200分子筛层析纯化后,得到目的 蛋白TTE1925.结果 纯化后蛋白的纯度达到99%以上.蛋白采用悬滴气相扩散法得到衍射至1.9(A)的棒状晶体.结论 成功制备了高纯度TTE1925蛋白,获得TTE1925蛋白质晶体,为进一步解析变旋酶的三维结构及其生物学功能分子机制研究奠定了基础.

Expression, Purification, and Crystallization of A Novel Galactose Mutarotase from Thermoanaerobacter Tengcongensis

Objective To purify a novel galactose mutarotase (TTE1925) from Thermoanaerobacter tengcongensis for crystallization and X-ray diffraction. MethodsThe tte 1925 gene was subcloned into the prokaryotic expression vector pGEX-6P-1 and overexpression was obtained in the E.coli BL21 (DE3) through transformation of the right recombinant plasmid that had been verified by colony PCR and sequencing. Soluble fusion protein with glutathione S-transferase tag expressed highly by the induction of isopropyl β-D-thiogalactoside and was purified in a three-step procedure, which included Glutathione Sepharose~(TM)4B affinity, ion chromatography (Resource Q 6mL), and gel filtration chromatography (10/300 superdex 200). ResultThe purity of the purified protein was over 99% and a large amount of claval crystals whose X-ray diffraction reached 1.9(A)were obtained. ConclusionsWe successfully prepared TTE1925 with high purity and obtained crystals for X-ray diffraction. These work paved the way for the further research on the 3-D structure of TTE1925 and its biological mechanism.