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

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+。%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+.