[1]徐朋奇),李杰),张红梅),等.杜氏盐藻类驱动蛋白钙调素结合蛋白马达区的表达与纯化*[J].郑州大学学报(医学版),2011,(06):831.
 XU Pengqi,LI Jie,ZHANG Hongmei,et al.Expression and purification of KCBP motor domain from Dunaliella salina[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2011,(06):831.
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杜氏盐藻类驱动蛋白钙调素结合蛋白马达区的表达与纯化*
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《郑州大学学报(医学版)》[ISSN:1671-6825/CN:41-1340/R]

卷:
期数:
2011年06期
页码:
831
栏目:
专题研究
出版日期:
2011-11-20

文章信息/Info

Title:
Expression and purification of KCBP motor domain from Dunaliella salina
作者:
徐朋奇1)李杰1)张红梅2)石科1)韩康1)李梅2)薛乐勋1)#
1)郑州大学生物工程系细胞生物学研究室 郑州 450001 2)中国科学院生物物理研究所生物大分子国家重点实验室 北京 100101
Author(s):
XU Pengqi1) LI Jie1) ZHANG Hongmei2) SHI Ke1)HAN Kang1) LI Mei2) XUE Lexun1)
1)Laboratory for Cell Biology,Department of Bioengineering,Zhengzhou University,Zhengzhou 450001 2)National Laboratory of Biomacromolecules,Institute of Biophysics,Chinese Academy of Sciences,Beijing 100101
关键词:
杜氏盐藻类驱动蛋白钙调素结合蛋白纯化
Keywords:
Dunaliella salinakinesinlike calmodulinbinding proteinpurification
分类号:
Q781
摘要:
目的:构建杜氏盐藻类驱动蛋白钙调素结合蛋白马达区(KMD)基因原核表达载体并表达纯化KMD。方法:通过软件和在线工具预测KMD的理化性质。构建KMD的原核表达载体pET28aKMD,而后在大肠杆菌BL21(DE3)内16 ℃过夜诱导表达KMD,通过超声破碎菌体和高速离心,将上清液进行亲和层析,获得粗纯目的蛋白,再利用离子交换层析和凝胶过滤层析进一步纯化。SDSPAGE检测层析各阶段蛋白样品。将纯化后的KMD均分3份分别放置在-80 ℃、4 ℃和18 ℃环境中,1周后SDSPAGE检测目的蛋白是否降解。结果:KMD相对分子质量为37 480,预测pI为7.99。基因中含有的稀有密码子不会明显影响其在大肠杆菌BL21(DE3)中表达。多步层析后可以获得2个独立的目的蛋白洗脱峰,均为KMD且纯度可满足结晶条件搜索。纯化后的KMD在4 ℃和18 ℃下放置1周后无降解。结论:获得大量高纯度、均一性良好的KMD蛋白,能够满足培养蛋白晶体所需。
Abstract:
Aim: To express and purify the kinesinlike calmodulinbinding protein motor domain (KMD) of Dunaliella salina by pET28a prokaryotic expression system and ion exchange and gel filtration chromatography.Methods:KMD cDNA was subcloned into the pET28a vector after physicochemical property of KMD was analyzed using software and online tool. The supernatant expressing the recombinant KMD proteins was collected by using sonication and highspeed centrifuge,and then the purified proteins were obtained after affinity chromatograph, ion exchange chromatograph and gel filtration chromatograph.The proteins obtained from each step were evaluated by SDSPAGE and kept at -80 ℃,4 ℃ and 18 ℃ for a week,respectively.Results: KMD proteins with the relative molecular weigh of 37 480 and pI of 7.99 were effectively expressed in BL21(DE3) regardless of its rare codon. After the multistep chromatograph,two independent eluting peaks were shown,from which the KMD proteins were collected with high purity and stable quality after they were placed at 4 ℃ and 18 ℃ for a week.Conclusion: Highly purified KMD has been obtained,which provides the qualified material in resolving KMD crystal structure.

参考文献/References:

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备注/Memo

备注/Memo:
*国家自然科学基金资助项目30700014;2009~2010年生物大分子国家重点实验室开放课题 #通讯作者,男,1944年2月生,教授,博士研究生导师,研究方向:肿瘤标志物与基因工程,Email:xuelx@zzu.edu.cn
更新日期/Last Update: 1900-01-01