Abstract:

To produce mouse metallothionein_Ⅰ (mMT_Ⅰ) in cyanobacterium Anabaena sp. PCC 7120, a novel Escherichia coli_cyanobacterium shuttle fusion expression vector, pKG_MT, was constructed. Via this vector, mMT_Ⅰ cDNA which was fused with a carboxyl terminal extension of the 26 kD glutathione_S_transferase (GST) containing a thrombin specific site was expressed in Anabaena under the control of tac promoter. SDS_polyacrylamid gel electrophoresis (SDS_PAGE) showed that the fusion protein GST_MT was expressed in the transgenic Anabaena sp. PCC 7120 after induction with isopropylthio_β_D_galactoside (IPTG). Glutatione_S_transferase metallothionein (GST_MT) was purified from the crude extracts by affinity chromatography on immobilized glutathione and mMT_Ⅰ was obtained by digesting the fusion protein with thrombin on column and gel filtration on Sephadex G_50. SDS_PAGE demonstrated that the purified mMT_Ⅰ was the desired protein. The result of ELISA for the purified mMT_Ⅰ showed that the recovery of mMT_Ⅰ from the transgenic cyanobacterium was about 0.6 mg/g fresh weight. According to the data of atomic absorption assay, metal_binding activity of the purified mMT_Ⅰ was almost the same as that of wild type MT.

鼠金属硫蛋白-I在鱼腥藻7120中的融合表达及其纯化
周杰 郝福英 施定基 俞梅敏 茹炳根
北京大学生命科学学院蛋白质工程及植物基因工程国家重点实验室 北京大学生命科学学院蛋白质工程及植物基因工程国家重点实验室 中国科学院植物研究所 北京大学生命科学学院蛋白质工程及植物基因工程国家重点实验室 北京大学生命科学学院蛋白质工程及植物基因工程国家重点实验室

【摘要】：为了提高小鼠金属硫蛋白-I(mMT-I)在鱼腥藻7120(Anabaena sp.PCC 7120)中的表达量、便于表达产物的分离纯化,构建了新的穿梭融合表达载体pKG-MT。通过pKG-MT,mMT-I cDNA在tac启动子的调控下,以与谷胱甘肽转硫酶(GST)C-末端相融合(GST-MT)的形式在鱼藻中表达。SDS-PAGE结果显示在异丙基硫代-β-D-半乳糖苷(IPTG)诱导下GST-MT在鱼腥藻中表达。经谷胱甘肽亲合层析,从转基因藻中分离、纯化得到GST-MT,利用GSTC-末端的凝血酶酶切位点,用凝血酶对GST-MT进行柱上酶切,经Sephadex G50除去凝血酶得到mMT-I。
【关键词】： 小鼠金属硫蛋白-Ⅰ 鱼腥藻 融合表达 亲合层析
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