Cao Xiaomin, Li Bing, Chen Zhen, Tu Hongbin, Fu Xin
Experimental Medical Research Center, Guangzhou Medical College, Guangzhou 510182, China.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2007 Apr;24(2):409-12, 419.
Metallothioneins (MT) are potential candidates for medicine development and application. For the purpose of expressing recombinant MT in E. coli, a crab MT cDNA cloned into pGEM-T was subcloned into pET-GST and then transformed into Escherichia Coli BL21. The fusion protein was proved to be expressed in both soluble and insoluble form by SDS-PAGE and western blot. Since metallothionein chelate metal ions, which may effects the physiological process of E. coli, caused the production of recombinant protein was lower than expected. Optimization of the ions content in the culture medium improved expression. The protein was purified by Zn2+ affinity chromatography, and rinsed off with high imidazole (1.5 M) which was the result of MT chelating instead of His-tag. This fusion protein laid a foundation of further study on the structural and functional biology of metallothionein.
金属硫蛋白（MT）是药物开发和应用的潜在候选物。为了在大肠杆菌中表达重组MT，将克隆到pGEM-T中的蟹MT cDNA亚克隆到pET-GST中，然后转化到大肠杆菌BL21中。通过SDS-PAGE和蛋白质印迹证明融合蛋白以可溶和不溶形式表达。由于金属硫蛋白螯合金属离子，这可能影响大肠杆菌的生理过程，导致重组蛋白的产量低于预期。优化培养基中的离子含量可提高表达。该蛋白通过Zn2+亲和层析纯化，并用高浓度咪唑（1.5 M）洗脱，这是MT螯合而非His标签的结果。这种融合蛋白为金属硫蛋白的结构和功能生物学的进一步研究奠定了基础。
