白念珠菌翻译起始因子蛋白Ca-tif1的表达和纯化

Expression and purification of translation initiation Ca-tif1 in Candida albican

本文构建了白色念珠菌体内翻译起始因子Ca-tif1的原核表达载体,纯化Ca-tif1蛋白,并测定其ATP酶活性。采用化学合成Ca-tif1序列片段,酶切并与p ET28a载体对应位点连接,连接后载体转化BL21感受态细胞,IPTG诱导Ca-tif1表达,用镍离子亲和层析柱纯化目的蛋白,并采用孔雀绿-磷钼酸分光光度法测定目的蛋白ATP酶活性。结果显示,根据大肠杆菌密码子偏好性进行密码子优化的重组质粒p ET-28a-Ca-tif1顺利表达可溶性的目的蛋白Ca-tif1,每升大肠杆菌表达产物经纯化后得到Ca-tif1 7.0 mg,纯化后的Ca-tif1具有ATP酶活性,1.25×106U/mg。通过成功构建白色念珠菌翻译起始因子Ca-tif1的原核表达载体p ET-28a-Ca-tif1,高效表达并纯化了Ca-tif1,经测定Ca-tif1具有ATP酶活性,为基于白色念珠菌翻译起始因子的一系列研究奠定了基础。

In order to construct the prokaryotic expression vector of Ca-tif1 and to purify the Ca-tif1,and to determine the ATPase activity of Ca-tif1,the gene Ca-tif1 gene was synthesized,and was inserted into pEt-28a,then was transformed into BL21;it was induced expression by IPTG and the target protein was purified by Ni ion affinity chromatography column.The ATP enzyme activity was determined by malachite green-phosphorus molybdenum acid spectrophotometry.After optimized codon according to the codon preference of E.coli,the target protein Ca-tif1was successfully expressed in soluble form in BL21.About 7 mg of expression product,with a purity of more than 95%,was obtained from each liter of E.coli.The purified Ca-tif1 had ATPase activity.The prokaryotic expression vector pEt-28a-Ca-tif1 of translation initiation factor Ca-tif1 in Candida albicans was successfully constructed.The Ca-tif1 was highly expressed and purified,and the activity of ATPase was determined,which laid a foundation for a series of studies based on the translation initiation factor in Candida albicans.