生物技术通报 ›› 2016, Vol. 32 ›› Issue (4): 121-127.doi: 10.13560/j.cnki.biotech.bull.1985.2016.04.016

• 研究报告 • 上一篇    下一篇

盐穗木金属硫蛋白HcMT在大肠杆菌中高效表达及金属离子结合能力的检测

孟红恩,杜荣俸,徐鑫,刘忠渊   

  1. 新疆大学生命科学与技术学院 新疆生物资源基因工程重点实验室,乌鲁木齐 830046
  • 收稿日期:2015-07-28 出版日期:2016-04-25 发布日期:2016-04-26
  • 作者简介:孟红恩,男,硕士研究生,研究方向:生物化学与分子生物学;E-mail:menghongenxju@163.com
  • 基金资助:
    新疆维吾尔自治区高技术研究发展项目(201416102)

High-level Expression of a Type 2 Metallothionein Protein(HcMT)from Halostachys caspica in Escherichia coli and Its Metal-binding Ability

MENG Hong-en, DU Rong-feng, XU Xin,LIU Zhong-yuan   

  1. Xinjiang Key Laboratory of Biological Resources and Genetic Engineering,College of Life Science and Technology, Xinjiang University,Urumqi 830046
  • Received:2015-07-28 Published:2016-04-25 Online:2016-04-26

摘要: 旨在研究盐穗木金属硫蛋白HcMT基因原核表达情况及结合金属离子的能力。从盐穗木中克隆获得的金属硫蛋白(HcMT)基因亚克隆至pGEX-6p-2原核表达载体上,在大肠杆菌BL21中表达,分离纯化融白蛋白GST-HcMT,通过原子吸收分光光度法测定融合蛋白GST-HcMT结合金属离子的量以判断其与金属离子的结合能力。结果显示,诱导表达的融合蛋白GST-HcMT分子量在34 kD左右,与预期一致,且通过Western blot进一步得到验证;原子吸收结果表明其具有结合Cu2+、Zn2+、Pb2+、Cd2+的能力,融合蛋白结合Cu2+、Zn2+、Pb2+、Cd2+的量分别是对照的25倍、10倍、4倍和2倍,其结合能力大小为Cu2+>Cd2+>Zn2+>Pb2+。

关键词: 盐穗木金属硫蛋白, 原子吸收分光光度法, 金属离子结合能力

Abstract: This study aims to evaluate the prokaryotic expression and metal-binding ability of metallothionein protein in Halostachys caspica(HcMT). HcMT cloned from H. caspica was transferred to prokaryotic expression vector pGEX-6p-2 and expressed in Escherichia coli BL21. Then the glutathione S-transferase(GST)fusion protein(GST-HcMT)was isolated and purified. Using atomic absorption spectrometry,the metal-binding ability of the GST-HcMT was determined by measuring the amount of metal ions it bound with. The results showed that the molecular weight of expressed fusion protein GST-HcMT was about 34 kD,which was as expected and confirmed by Western blot. Furthermore,GST-HcMT bound 25,10,4,2 folds of Cu2+,Zn2+,Pb2+,and Cd2+ separately as GST alone,and the binding ability of GST-HcMT was as follows:Cu2+ > Cd2+ > Zn2+ > Pb2+.

Key words: metallothionein protein from Halostachys caspica(HcMT), atomic absorption spectrometry(AAS), metal-binding ability