食品科学 ›› 2010, Vol. 31 ›› Issue (3): 177-181.doi: 10.7506/spkx1002-6300-201003040

• 生物工程 • 上一篇    下一篇

玉米谷氨酰胺转胺酶基因的克隆及原核表达

秦兰霞1,王 利2,张兰威3,*   

  1. 1.东北农业大学 黑龙江省乳品工业技术开发中心 2.完达山乳业股份有限公司 3.哈尔滨工业大学食品科学与工程学院
  • 收稿日期:2008-11-12 修回日期:2009-09-25 出版日期:2010-02-01 发布日期:2010-12-29
  • 通讯作者: 张兰威 E-mail:zhanglw@hit.edu.cn,lanweizhang@yahoo.com.cn
  • 基金资助:

    “十一五”国家科技支撑计划项目(2006BAD04A09) 黑龙江省自然科学基金项目(ZJNO605-02) 黑龙江省“十一五”科技攻关资助项目(GAO7B401) 国家自然科学基金项目(30972041/C110602)

Cloning and Expression of Maize Transglutaminase in Escherichia coli

QIN Lan-xia1 WANG Li2 ZHANG Lan-wei3,*   

  1. 1. Heilongjiang Dairy Industry Technical Development Center, Northeast Agricultural University, Harbin 150086, China
    2. Wondersun Dairy Co. Ltd., Harbin 150078, China
    3. School of Food Science and Engineering, Harbin Institute of Technology, Harbin 150090, China
  • Received:2008-11-12 Revised:2009-09-25 Online:2010-02-01 Published:2010-12-29
  • Contact: ZHANG Lan-wei E-mail:zhanglw@hit.edu.cn,lanweizhang@yahoo.com.cn

摘要:

从玉米嫩叶中提取总RNA,通过RT-PCR的方法扩增出玉米谷氨酰胺转胺酶(TGase)全长基因,回收目的片段并测序,该基因编码区全长1605bp,编码535个氨基酸残基,分子量为60.9kD,与GenBank(登录号:AJ421525)上已发表的序列同源性为100%。按正确的阅读框架将玉米TGase基因片段定向克隆到表达载体pET-28a上,将重组质粒转化到大肠杆菌Rosetta(DE3)菌株,1mmol/L IPTG诱导融合蛋白表达,经凝胶分析软件测得蛋白表达量约占总蛋白的15%,以His-Tag抗体作为一抗,采用Western-blot方法检测目的蛋白,结果证明所表达的特异蛋白是带有His-Tag的重组融合蛋白,利用Ni2+-NTA琼脂糖树脂亲和层析柱纯化目的蛋白,SDS-PAGE鉴定为单一条带,测得纯化后的TGase酶活力达到16U/mg。

关键词: 玉米嫩叶, 大肠杆菌, 谷氨酰胺转胺酶, 表达, 酶活

Abstract:

In this study, total RNA was extracted from young leaves of maize. Reverse transcription PCR (RT-PCR) method was used to obtain full-length transglutaminase (TGase) gene. The amplified fragment was sequenced to have 1605 bp. The gene consisted of 535 amino acid residues with a calculated molecular mass of 60.9 kD. It was identical to the published TGase gene (GenBank NO. AJ421525). This gene fragment was cloned into pET-28a expression vector. The pET-28a-TGase was transformed into E. coli Rosetta (DE3) and expressed in E. coli cells with the induction of 1 mmol/L IPTG. According to quantitative analysis, the expression amount of this protein was approximately 15% of total protein. Western-blot analysis confirmed that this protein was a His-tag recombinant protein. After the purification by Ni2+-NTA affinity chromatography, the purified protein exhibited high purity and one single band was shown in SDS-PAGE. The TGase activity was up to 16 U/mg after purification.

Key words: maize, E. coli, transglutaminase, expression, activity

中图分类号: