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OALib Journal期刊
ISSN: 2333-9721
费用：99美元
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大肠杆菌系列融合表达载体的构建
, PP. 97-102
Keywords: 融合表达载体,融合标签,伴侣蛋白,tev,n-乙酰-d-葡萄糖胺2-异构酶
Abstract:
许多异源蛋白在大肠杆菌内的表达是以不可溶、无生物学活性的包涵体形式存在,这为蛋白质的功能研究带来困难.融合表达是提高蛋白可溶性的有效方案之一.为构建通用型融合表达载体,本研究将5种常见的融合标签即突变型麦芽糖结合蛋白(mmbp)、小分子泛素样修饰蛋白(sumo)、翻译起始因子(if2-i)、氮源利用物质a(nusa)和谷胱甘肽转移酶(gst)以及3种伴侣蛋白(groel、dnak和tf)分别克隆至pet30a(+),构建了系列融合表达载体.这些载体含有相同的克隆位点以及位于融合标签羧基端的烟草蚀刻病毒蛋白酶(tev)的酶切位点.n-乙酰-d-葡萄糖胺2-异构酶基因hrnbp克隆到mmbp融合表达载体后,经iptg诱导,sds-page检测表明融合蛋白均得到了高效表达且几乎完全可溶,tev酶切获得了预期的带型.全细胞偶联生成n-乙酰-d-神经氨酸实验发现mmbp-hrnbp的摩尔转化率较无mmbp标签的体系提高了近60%,证明了融合表达载体中融合标签的适用性.新型的原核融合表达载体为蛋白的融合表达、分离纯化及功能研究提供了更多的选择.
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