在线阅读 --自然科学版 2009年1期《rPA(K)原核表达产物的复性与纯化》
rPA(K)原核表达产物的复性与纯化--[在线阅读]
罗惠霞, 李敏, 王玉炯, 田楠
宁夏大学生命科学学院重点实验室, 宁夏银川750021
起止页码: 100--106页
DOI:
摘要
为探索新型rPA(K)在原核系统中表达产物的纯化和复性,采用不同的洗涤剂(脲、Triton X-100、乙醇)依次洗涤包涵体,又经硫酸铵沉淀,得到了经初纯后纯度较高的包涵体.结果表明,经2 mol/L脲1、mL/L TritonX-100、200 mL/L乙醇洗涤,15%饱和度的硫酸铵沉淀后,包涵体的纯度由16.0%提高到56.6%.在此基础上,利用稀释复性和透析复性2种方法对包涵体进行复性,以恢复产物的天然构象,获得具有生物学活性的目的蛋白.结果如下:采用透析复性法,rPA(K)的比活可达1.33×105IU/mg;采用稀释复性法,rPA(K)的比活可达1.06×105IU/mg.利用S.TagTMrEK纯化试剂盒对目的产物进行进一步纯化,并对rEK和MgCl22种洗脱剂的洗脱效果进行了比较.SDS-PAGE结果显示,MgCl2洗脱后的产物,分子量未发生变化,仍为43 ku,其纯度为72%;而rEK洗脱后的产物,其纯度达到了72%,且分子量减小至约40 ku,表明去除了融合蛋白中的非目的片段.

The Purification and Renaturation of rPA(K) Prokaryotic Expressive Product
LUO Hui-xia, LI Min, WANG Yu-jiong, TIAN Nan
College of Life Science, Ningxia University, Ningxia Yinchuan 750021, China
Abstract:
In order to study the purification and renaturation of rPA(K) inclusion body in the prokaryotic system,the dialysis renaturation and dilution renaturation was used to renature the target product respectively.Through dialysis renaturation,the specific activity was 1.33×105 IU/mg;while it was 1.06×105 IU/mg by using dilution renaturation.The renatured rPA(K) was further purified by S·TagTM rEK purification kit,and the two kinds of eluent(rEK and MgCl2) were compared.SDS-PAGE indicated that the molecular weight of target protein had no change by using MgCl2 and was still 43 ku.While,by using rEK,the molecular weight of it was 40 ku.That meant the non-target fragment was cut off,and the purity of product reached 72%.

收稿日期: 2008-6-11
基金项目: 宁夏自然科学基金(NZ0505)

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