Prokaryotic expression, purification and identification of human mutant nm23-H1 protein

YANG Xue-qin; et al

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Prokaryotic expression, purification and identification of human mutant nm23-H1 protein

更新时间：2024-01-17

- Prokaryotic expression, purification and identification of human mutant nm23-H1 protein
- Journal of Chongqing Medical University Vol. 34, Issue 9, Pages: 1235-1238(2009)
- 作者机构：
  
  1. 第三军医大学大坪医院野战外科研究所肿瘤中心
  2. 天津医科大学总医院天津市肺癌研究所
- 作者简介：
- 基金信息：
- DOI：
  CLC： Q78
- Published：2009，
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[1]杨雪琴,周清华,王东,王阁,马力,朱大兴.人重组蛋白nm23-H1突变体原核表达纯化与鉴定[J].重庆医科大学学报,2009,34(09):1235-1238.

YANG Xue-qin, et al. Prokaryotic expression, purification and identification of human mutant nm23-H1 protein. [J]. Journal of Chongqing Medical University 34(9):1235-1238(2009)
[1]杨雪琴,周清华,王东,王阁,马力,朱大兴.人重组蛋白nm23-H1突变体原核表达纯化与鉴定[J].重庆医科大学学报,2009,34(09):1235-1238. DOI：

YANG Xue-qin, et al. Prokaryotic expression, purification and identification of human mutant nm23-H1 protein. [J]. Journal of Chongqing Medical University 34(9):1235-1238(2009) DOI：

摘要

目的:构建nm23-H1的S120G

S120G-P96S及S44A突变体原核表达载体

并在大肠杆菌中表达及纯化。方法:通过PCR方法扩增突变型nm23-H1目的片段

利用基因重组技术构建pET28a-nm23-H1突变型原核表达载体

并进行酶切和测序鉴定

鉴定正确克隆转化BL21（DE3）溶源菌

进行蛋白表达及可溶性分析

同时采用NI-NTA镍柱亲和层析法进行纯化

应用Westernblot方法对纯化后蛋白进行鉴定检测。结果:通过酶切和测序鉴定

pET28a-nm23-H1突变型原核表达载体序列正确

编码框正确。转化溶源菌后能够表达目的蛋白

蛋白表达量高;其中

S44A为可溶性表达

S120G以可溶性表达为主

少部分为包涵体表达

而S120G-P96S则大部分为包涵体表达;Westernblot检测结果提示3个突变型nm23-H1蛋白分子量均为20kD

与预计值相符。结论:成功构建nm23-H1的S120G

S120G-P96S及S44A突变体原核表达载体

S120G和S44A蛋白可以用于下一步实验研究

而S120G-P96S突变体则可能不适于下一步研究。

Abstract

Objective:To construct prokaryotic expression vector of mutant nm23-H1(S120G

S120G-P96S

S44A)

and then express and purify the proteins. Methods: Mutant nm23-H1 fragments were amplified by PCR. The prokaryotic expression vectors of pET28a-nm23-H1 were constructed by gene recombination technique and verified by restriction enzyme analysis and sequencing. The positive clones were transformed into E. coli BL21(DE3) and soluble analysis of the expression was conducted in these systems. The proteins were purified by nickel column chromatography and identified by Western blot. Results: The sequences and open read frames of all the pET28a-nm23-H1 were completely correct. After transforming

these plasmids could express the target proteins. The protein production was very high. Among these mutant proteins

S44A was soluble expression and S120G was partly soluble. However

S120G-P96S was mostly in the inclusion bodies. The molecular weight of these mutant proteins was 20 kD detected by Western blot

which was as the same as the objective protein. Conclusion: We have succeeded in constructing the prokaryotic expression vectors of pET28a-nm23-H1(S120G

S120G-P96S

S44A)

and the S120G and S44A proteins can be used in following studies. However

S120G-P96S protein which expressed in this system may not be suitable for the following studies.

关键词

nm23-H1突变原核表达

Keywords

Nm23-H1mutationprokaryotic expression

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