大肠杆菌糖基转移酶WclR蛋白的表达纯化及功能鉴定
Expression, puri?cation, and characterization of GalactosyltransferaseWclR from Escherichia coli
构建WclR的蛋白表达载体，蛋白表达纯化，并进行功能鉴定。首先利用PCR扩增大肠杆菌O3上的wclR基因，酶切连接到pGEX4T-1载体上，成功构建pGEX4T-1-wclR表达载体，转化至E.coli BL21(DE3)菌株，低温下IPTG诱导蛋白表达，利用GST亲和层析柱进行蛋白纯化，Western blot进行蛋白验证。利用UDP-Gal为糖基供体，合成GlcNAc-a-PO3-PO3-(CH2)9-CH3 (GlcNAc-PP-De)为受体进行酶活验证，质谱检测证明在WclR的作用下能成功的合成Gal-GlcNAc-PP-De。实验结果证明WclR具有半乳糖糖基转移酶活性。?????
In this paper we report expression and puri?cation of WclR protein, the function was identified. The expression of WclR at a low temperature significantly increased the amount of the soluble portion of the overall protein. The plasmid pGEX-4T-1 was used to express WclR with a GST affinity tag. The protein was confirmed as WclR by western blotting. The enzymatic product was identified by mass spectrometry using UDP-Gal as the donor substrate and the synthetic acceptor substrate GlcNAc-a-PO3-PO3-(CH2)9-CH3 (GlcNAc-PP-De). This study confirmed thatWclR was a galactosyltransferase (GalT) that acts on the linkage Gal -GlcNAc.
陈超、刘斌、冯露
生物化学分子生物学微生物学
大肠杆菌蛋白纯化wclR糖基转移酶
Escherichia coliPuri?cationWclRGlycosyltransferases
陈超,刘斌,冯露.大肠杆菌糖基转移酶WclR蛋白的表达纯化及功能鉴定[EB/OL].(2015-12-30)[2025-07-23].http://www.paper.edu.cn/releasepaper/content/201512-1367.点此复制
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