[1]李素梅,卢颖洪,周敏.利用PCR介导的同源重组制备V型ATP酶的亚基蛋白VPH1[J].江苏农业学报,2017,(05):1016-1021.[doi:doi:10.3969/j.issn.1000-4440.2017.05.009]
　LI Su-mei,LU Ying-hong,ZHOU Min.Preparation of VPH1 protein of V-ATPase using PCR-mediated homologous recombination[J].,2017,(05):1016-1021.[doi:doi:10.3969/j.issn.1000-4440.2017.05.009]
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利用PCR介导的同源重组制备V型ATP酶的亚基蛋白VPH1()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2017年05期
页码:
1016-1021
栏目:
遗传育种·生理生化
出版日期:
2017-10-30

文章信息/Info

Title:
Preparation of VPH1 protein of V-ATPase using PCR-mediated homologous recombination
作者:
李素梅; 卢颖洪; 周敏
（南京理工大学化工学院，江苏南京210094）
Author(s):
LI Su-mei; LU Ying-hong; ZHOU Min
（School of Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, China）
关键词:
PCR介导; 同源重组; V型ATP酶; VPH1蛋白制备
Keywords:
polymerase chain reaction（PCR）-mediation; homologous recombination; V-ATPase; VPH1 preparation
分类号:
Q784
DOI:
doi:10.3969/j.issn.1000-4440.2017.05.009
文献标志码:
A
摘要:
V型三磷酸腺苷酶（ATP酶）是真核生物调节胞质内细胞器酸碱度的核心元件，对盐等胁迫环境下细胞膜的稳定起着重要作用。VPH1是V型ATP酶蛋白复合物膜上的1个亚基蛋白，其跨膜结构对V型ATP酶的功能有很大影响。本研究利用PCR（Polymerase chain reaction）介导的同源重组技术，构建毕赤酵母内源VPH1 C端的定点融合FLAG标签，筛选重组菌株进行培养，分离出目的蛋白，去污剂溶解后通过FLAG亲和纯化获得VPH1蛋白，为其后续结构的研究奠定基础。说明，PCR介导的同源重组技术能快速筛选到毕赤酵母重组转化子，FLAG标签添加到VPH1的C端可以成功纯化出目的蛋白。同时本研究所用的载体和研究思路也可以用于其他感兴趣膜蛋白的重组纯化。
Abstract:
V-ATPase, the core component of eukaryotes to adjust pH of cytoplasmic organelles, plays an important role in cell membrane stability under stress environment. VPH1 is one subunit of V-ATPase membrane-bound Vo protein complex and its structure decides the function of V-ATPase to a large extent. PCR-mediated DNA homologous recombination transformation was adopted to tag C-terminus of P. pastoris VPH1. The screened recombinant strain was cultured for isolating target protein. Subsequently detergent dissolving was applied to anti-FLAG agarose for VPH1 purification. The results showed that the VPH1 protein from P. pastoris was successfully isolated using a fusion FLAG tag attached to the C-terminus of VPH1.

参考文献/References:

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备注/Memo

备注/Memo:
收稿日期：2017-04-08 基金项目：国家自然科学基金项目（21375062） 作者简介：李素梅（1978-），女，安徽合肥人，博士，助理研究员，主要从事膜蛋白制备研究。(E-mail) smli321@163.com 通讯作者：周敏，(E-mail)minzhou@njust.edu.cn
更新日期/Last Update: 2017-11-03