白念珠菌HSP90与人源HSP90的克隆表达及其C端的比较分析

中国真菌学杂志　 2016, Vol. 11 　Issue (6): 321-326.

论著下一篇

白念珠菌HSP90与人源HSP90的克隆表达及其C端的比较分析

孟靖顺¹, 吴建华¹, 赵静¹, 王玉莲¹, 薛伟²

1. 上海长海医院, 上海 200433;
2. 中国科学院大学, 上海 200031

收稿日期:2016-09-28 出版日期:2016-12-28 发布日期:2016-12-28
通讯作者: 吴建华,E-mail:wujh_cn@163.com E-mail:wujh_cn@163.com
作者简介:孟靖顺,男(汉族),硕士研究生在读.E-mail:1781069890@qq.com
基金资助:
973子课题（2013CB531602）

Purification and C-terminal domain comparison of heat shock protein 90(HSP90) in Candida albicans and human

MENG Jing-shun¹, WU Jian-hua¹, ZHAO Jing¹, WANG Yu-lian¹, XUE Wei²

1. Changhai hospital, Shanghai 200433, China;
2. University of Chinese Academy of Sciences, Shanghai 200031, China

Received:2016-09-28 Online:2016-12-28 Published:2016-12-28

摘要/Abstract

摘要：

目的分别体外表达纯化白念珠菌HSP90（CaHSP90）和人源HSP90（hHSP90）并分析C-端序列差异对其体外状态下分子聚合度的影响。方法我们将CaHSP90、hHSP90野生型及其C-端酶切碱基序列通过PCR进行扩增并通过NdeI/XhoI酶切位点连接到到pET22b⁺质粒中。将构建好的上述质粒转入大肠杆菌E. coli BL21（DE3）中进行表达并通过Ni²⁺-NTA柱以及Superdex-200快速柱层析系统（FPLC）纯化。所得蛋白通过SDS-PAGE以及分子排阻色谱（SEC）测定分子量和聚合状态。结果通过测序鉴定所有质粒构建成功，并最终纯化得到足够纯度的蛋白进行SEC分析实验。分析结果表明野生型CaHSP90和hHSP90在体外具有相似的聚合度，而C-端突变后则存在明显差异。结论 CaHSP90和hHSP90的C-端结构域对其体外聚合状态有着不同的影响，CaHSP90的C-端结构域有可能成为CaHSP90抑制剂一个潜在靶点。

关键词: 白念珠菌, HSP90, 蛋白结构, 蛋白表达与纯化

Abstract:

Objective To express and purify Candida albicans HSP90 (CaHSP90) and human HSP90 (hHSP90) and to compare the different effects of C-terminal domain on their oligomeric forms in vitro.Method The encoding sequences for wild type CaHSP90,hHSP90 and their C-terminal enzyme digested base sequences were amplified via PCR and the products were cloned into pET-22b⁺ vector using NdeI/XhoI cloning sites.The plasmids were expressed in E. coli BL21 (DE3) cells and the His-tagged proteins were purified through Ni²⁺-NTA column and Superdex-200 column chromatography on FPLC. The molecular weights and oligomeric forms of these proteins were analyzed by SDS-PAGE and size exclusion chromatography (SEC).Results All plasmids were successfully constructed by DNA sequencing and the proteins were pure enough for SEC analysis.The results showed that wild type CaHSP90 and hHSP90 have similar oligomeric forms,while their C-terminal mutants were different.Conclusion The C-terminal domain of CaHSP90 and hHSP90 have different effect on their oligomeric forms in vitro and might be a potential target for CaHSP90 inhibitor.

Key words: Candida albicans, HSP90, protein structure, protein expression and purification

中图分类号:

R379.4

孟靖顺, 吴建华, 赵静, 王玉莲, 薛伟. 白念珠菌HSP90与人源HSP90的克隆表达及其C端的比较分析[J]. 中国真菌学杂志, 2016, 11(6): 321-326.

MENG Jing-shun, WU Jian-hua, ZHAO Jing, WANG Yu-lian, XUE Wei. Purification and C-terminal domain comparison of heat shock protein 90(HSP90) in Candida albicans and human[J]. Chinese Journal of Mycology, 2016, 11(6): 321-326.

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白念珠菌HSP90与人源HSP90的克隆表达及其C端的比较分析

Purification and C-terminal domain comparison of heat shock protein 90(HSP90) in Candida albicans and human

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