摘要
通过PCR从酿酒酵母基因组DNA上扩增得到酿酒酵母基因CUP1编码的金属硫蛋白序列.将其编码序列克隆到质粒pTWIN1构建重组表达质粒pTWIN1-MT,转化大肠杆菌感受态细胞ER2566.重组融合蛋白CBD-intein1-MT在IPTG的诱导下得到表达,经SDS-PAGE和蛋白质印迹鉴定.用重组菌分别进行铜离子耐受性和吸收等实验,结果表明金属硫蛋白的表达增加了重组菌对铜离子的耐受性和积累量.用chitin亲和层析纯化重组蛋白,并由intein介导融合蛋白的自裂解.SDS-PAGE分析,纯化后的金属硫蛋白的纯度达到95%.纯化后的MT显示出很好的清除羟自由基的能力.
The gene fragment of metallothionein coded by gene CUP1 was amplified using PCR from Saccharomyces cerevisiae genome, and was cloned into plasmid pTWIN1 to construct the recombinant plasmid pTWIN1-CBD-MT. It was then transformed into the competent E. coli ER2566 cells. The recombinant fusion protein CBD-inteinl-MT was induced by IPTG and identified by SDS-PAGE and the western blot. The results of resistance and accumulation of copper ions using recombinant strain showed that the expression of metallothionein increased the tolerance and accumulation of Cu^2＋ in recombinant strain. The purification of recombinant protein was carried out by using chitin affinity chromatography and inteln-mediated self-cleavage. The purity of purified MT reached 95% and the purified MT showed strong ability to scavenge hydroxyl radicals.
出处
《兰州大学学报（自然科学版）》
CAS
CSCD
北大核心
2011年第3期58-62,67,共6页
Journal of Lanzhou University(Natural Sciences)
基金
珠海市科技攻关项目(PC20061065)
关键词
金属硫蛋白
酿酒酵母
原核表达
活性检测
metallothionein
Saccharomyces cerevisiae
prokaryotic expression
bioactivity analysis
