人SCYL1-BP1重组蛋白的原核表达及分离纯化鉴定

中国生物工程杂志

2012, Vol. 32

Issue (09): 1-8

研究报告

人SCYL1-BP1重组蛋白的原核表达及分离纯化鉴定

陈晓静, 陈小梅, 王洋, 施慧莉, 霍克克

复旦大学生命科学学院遗传工程国家重点实验室上海 200433

Prokaryotic Expression and Purification of Human SCYL1-BP1 and Its Identification

CHEN Xiao-jing, CHEN Xiao-mei, WANG Yang, SHI Hui-li, HUO Ke-ke

State Key Laboratory of Genetic Engineering, College of Life Sciences, Fudan University, Shanghai 200433, China

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摘要： 前期研究结果发现,SCYL1-BP1 具有细胞周期调控功能,同时具有肿瘤抑制因子的特性。目的:采用基因工程技术,构建SCYL1-BP1 的大肠杆菌重组表达菌株,以获得足够量的高纯度目的蛋白,为后面进行一系列药理学检测及新药安全性测试奠定基础。方法:利用从人胎脑cDNA文库中克隆得到SCYL1-BP1 基因克隆为模板,经PCR扩增,通过酶切位点克隆到新型原核表达载体pET-28b-SUMO上,转化大肠杆菌表达菌BL21(DE3)。经IPTG诱导表达,摸索优化表达条件,表达产物经Ni柱进行亲和层析纯化,后再进行SDS-PAGE和Western blot等分析鉴定。结果:成功构建了SCYL1-BP1 的原核表达工程菌BL21(DE3)/pET-28b-SUMO-SCYL1BP1。SDS-PAGE和Western blot检验结果表明,诱导表达的融合蛋白His6-SUMO-SCYL1BP1的分子量约为65 kDa,主要以可溶的形式存在,且能被His标签抗体和SCYL1-BP1单克隆抗体特异性识别。结论:原核表达并纯化了人SCYL1-BP1融合蛋白,为其后续功能研究及性质实验奠定基础。

关键词： SCYL1-BP1; 重组蛋白; 原核表达; 工程菌; SUMO

Abstract: Previous studies revealed that human SCYL1-BP1 gene plays an important role in the cell cycle and tumor suppression. Objective: Construction of recombinant human SCYL1-BP1 gene expression system in prokaryotic strain, in order to obtain sufficient purified SCYL1-BP1 protein for the pharmacology experiments.Methods: The full length coding sequence of SCYL1-BP1 was cloned from human placenta cDNA library by PCR and inserted into plasmid pET-28b-SUMO, resulted in the recombinant prokaryotic expression plasmid pET-28b-SUMO-SCYL1BP1. Then it was transformed into E. coli strains BL21(DE3) and Rosetta(DE3) for expression using IPTG as an inducer. The expressed recombinant protein was purified by Ni-NTA chromatography and identified by SDS-PAGE and Western blot with anti-His-tag and anti-SCYL1BP1 monoclonal antibody.Results: The recombinant prokaryotic expression strain BL21(DE3)-pET-28b-SUMO-SCYL1BP1 was constructed successfully. SDS-PAGE and Western blot results showed that the fusion protein was 65 kDa in soluble form, which can be recognized by anti-His-tag antibody and anti-SCYL1-BP1 monoclonal antibody.Conclusion: The recombinant human SCYL1-BP1 gene was successfully expressed in the E. coli strain BL21(DE3), and the purified SCYL1-BP1 protein was identified correctly by anti-SCYL1BP1 monoclonal antibody. The results provided a favorable condition for further study on SCYL1BP1 function and application.

Key words: SCYL1-BP1 Recombinant protein Prokaryotic expression Engineering bacteria SUMO

收稿日期: 2012-05-08 出版日期: 2012-09-25

ZTFLH:

Q786

基金资助: 国家"863"计划(2006AA02A310);国家科技重大专项项目(2008ZX10003－006,2009ZX09301-011);国家"973"计划(2010CB912602)资助项目

通讯作者: 霍克克 E-mail: kkhuo@fudan.edu.cn

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引用本文:

陈晓静, 陈小梅, 王洋, 施慧莉, 霍克克. 人SCYL1-BP1重组蛋白的原核表达及分离纯化鉴定[J]. 中国生物工程杂志, 2012, 32(09): 1-8.

CHEN Xiao-jing, CHEN Xiao-mei, WANG Yang, SHI Hui-li, HUO Ke-ke. Prokaryotic Expression and Purification of Human SCYL1-BP1 and Its Identification. China Biotechnology, 2012, 32(09): 1-8.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I09/1

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