PZR基因的定点突变、表达和纯化

中国生物工程杂志

2009, Vol. 29

Issue (03): 36-40

研究报告

PZR基因的定点突变、表达和纯化

莫毅^1,4,王伟²,梁方方³,杨旭芬²,宋茜²,蒋和生³,Wayne Zhou²

1. 广西大学动物科学技术学院 2. 美国路易斯安那州立大学生命科学院 3. 广西畜牧研究所 4.广西人口和计划生育研究中心

Site-directed Mutagenesis, Expression And Purification Of PZR

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摘要：

为获得纯化的单磷酸化PZR蛋白，使用双磷酸化位点的His-PZR重组原核表达载体为模板，采用重叠延长PCR 定点突变技术分别构建：PZRPhe263(TTT)和PZR-Phe241(TTT)重组原核表达载体，并与C-Src重组原核表达载体共同转化E.coli BL21菌株，通过IPTG诱导表达，Ni-NTA纯化，获得了单磷酸化的PZR a和PZR b蛋白，为进一步研究PZR内的不同ITIM基序与酪氨酸磷酸酶间的作用机制及其在信号转导过程中的功能奠定了基础。

关键词： PZR;免疫受体酪氨酸抑制基序;定点突变;表达;纯化

Abstract:

Objective In order to express and purify the mono-phosphorylated PZR proteins. Methods The bi-- phosphorylated His-PZR recombined prokaryotic expressions plasmids was used as the template, then site-directed mutagenesis was used to amplified the mono- phosphorylated PZR a Tyr263(TAT) Phe263(TTT)and PZR b Tyr241(TAT) Phe241(TTT) mutants. The mutants’ and C-Src recombined prokaryotic expressions plasmids were co-transformed to the E.Coli. BL21. Through IPTG induction and Ni-NTA purification, we want to get the mono-phosphorylated PZR a and PZR b proteins. Results The mono- phosphorylated PZR a and PZR b recombined prokaryotic expressions plasmids have successfully been constructed; The mutants have successfully been induced to express by IPTG. They represent 8.5% of the total bacterial protein in E.Coli. BL21, and theirs relative molecule mass is 10 000. After Ni-NTA purification, the mono-phosphorylated PZR a and PZR b proteins have been obtained. Conclusion These results laid a foundation to future study the bio-function mechanism between different ITIM of PZR with protein tyrosine phosphatase in the signal transduction.

Key words: PZR;ITIM;Site-directed mutation;Expression;Purification

收稿日期: 2008-10-13 出版日期: 2009-03-31

ZTFLH:

R392.11

通讯作者: Wayne Zhou

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	莫毅王伟梁方方杨旭芬宋茜蒋和生 Wayne Zhou

引用本文:

莫毅,王伟,梁方方,杨旭芬,宋茜,蒋和生,Wayne Zhou. PZR基因的定点突变、表达和纯化[J]. 中国生物工程杂志, 2009, 29(03): 36-40.

MO Yi- Wang-Wei- Liang-Fang-Fang- Yang-Xu-Fen- Song-Qian- Jiang-He-Sheng- Wayne Zhou. Site-directed Mutagenesis, Expression And Purification Of PZR. China Biotechnology, 2009, 29(03): 36-40.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I03/36

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