雷竹SOC1蛋白原核表达及纯化

doi:10.3969/j.issn.1000-2006.2016.03.007

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南京林业大学学报（自然科学版） ›› 2016, Vol. 40 ›› Issue (03): 41-46.doi: 10.3969/j.issn.1000-2006.2016.03.007

雷竹SOC1蛋白原核表达及纯化

汤叶根,施泉,林新春,徐英武^*

浙江农林大学,亚热带森林培育国家重点实验室培育基地,浙江临安 311300

出版日期:2016-06-18 发布日期:2016-06-18
基金资助:
收稿日期:2015-03-18 修回日期:2015-12-03
基金项目:国家自然科学基金项目(31270715,31000295); 浙江农林大学发展基金项目(2010FR072)
第一作者:汤叶根(735938916@qq.com)。*通信作者:徐英武(yxu@zafu.edu.cn),教授。
引文格式:汤叶根,施泉,林新春,等. 雷竹SOC1蛋白原核表达及纯化[J]. 南京林业大学学报(自然科学版),2016,40(3):41-46.

Expression and purification of Phyllostachys violascens SOC1 protein in prokaryotic system

TANG Yegen,SHI Quan, LIN Xinchun, XU Yingwu^*

The Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Lin'an 311300, China

Online:2016-06-18 Published:2016-06-18

摘要/Abstract

摘要： 为了获得有活性的雷竹PvSOC1蛋白(Phyllostachys violascens SUPPERSSOR OF CO OVEREXPRESSION 1),并进一步讨论其结构形态,通过原核表达方法得到PvSOC1的可溶性重组蛋白,以pET-GST作为表达载体,大肠杆菌E.coli Rosetta作为宿主菌株,超表达全长和IKC结构域的雷竹PvSOC1蛋白。发现在37℃条件下,菌液浓度D(600)值达到0.4～0.6时进行诱导,控制IPTG终浓度为0.4 mmol/L,诱导目的蛋白表达5 h,可以获得可溶的IKC结构域GST融合蛋白。采用TEV(tobacco etch virus protease)酶切技术、配合GST亲和层析柱及分子层析色谱柱,去除标签蛋白并纯化目的蛋白。所获得的高纯度IKC结构域PvSOC1蛋白经过对比分析发现其以八聚体形式存在。

Abstract: In order to obtain soluble recombinant PvSOC1(Phyllostachys violascens SUPPERSSOR OF CO OVEREXPRESSION 1)protein in vitro. We used pET-GST as expression vector, to over-express full-length and IKC structural domain of PvSOC1 in E.coli Rosetta prokaryotic system. We found the optimal expression strategy is when the suspension culture's OD₆₀₀ value reaches 0.4-0.6, add IPTG and keep it's concentration at 0.4 mmol/L. Kept incubating for 5 h at the temperature of 37℃ to induce the protein.We found IKC domain is soluble,to compare with the fullength sequence,it started from No.62 amino acid and ends at No.225. Added TEV(tobacco etch virus protease)protease to remove GST tag.Combined glutathione sepharose resin with size exclusion chromatography(SEC)to get highly purified IKC domain of PvSOC1.And chromatography data proof this domain exists as a octamer.

中图分类号:

Q518.2

汤叶根,施泉,林新春,等. 雷竹SOC1蛋白原核表达及纯化[J]. 南京林业大学学报（自然科学版）, 2016, 40(03): 41-46.

TANG Yegen,SHI Quan, LIN Xinchun, XU Yingwu. Expression and purification of Phyllostachys violascens SOC1 protein in prokaryotic system[J].Journal of Nanjing Forestry University (Natural Science Edition), 2016, 40(03): 41-46.DOI: 10.3969/j.issn.1000-2006.2016.03.007.

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雷竹SOC1蛋白原核表达及纯化

Expression and purification of Phyllostachys violascens SOC1 protein in prokaryotic system

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