CAJ | 학술논문

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 GS0除去凝血酶得到mMT-Ⅰ.SDS-PAGE表明纯化得到所要的目标产物;ELISA测定结果显示从每克转基因藻(鲜重)中可纯化得到0.9 mg mMT-Ⅰ;原子吸收测定表明纯化得到的mMT-Ⅰ的镉离子结合能力接近于天然MT.
위료제고소서금속류단백-Ⅰ(mMT-Ⅰ)재어성조7120(Anabaena sp.PCC 7120)중적표체량、편우표체산물적분리순화,구건료신적천사융합표체재체pKG-MT.통과pKG-MT,mMT-Ⅰ cDNA재tac계동자적조공하,이여곡광감태전류매(GST)C-말단상융합(GST-MT)적형식재어조중표체.SDS-PAGE결과현시재이병기류대-β-D-반유당감(IPTG)유도하GST-MT재어성조중표체.경곡광감태친합층석,종전기인조중분리、순화득도GST-MT.이용GSTC-말단적응혈매매절위점,용응혈매대GST-MT진행주상매절,경Sephadex GS0제거응혈매득도mMT-Ⅰ.SDS-PAGE표명순화득도소요적목표산물;ELISA측정결과현시종매극전기인조(선중)중가순화득도0.9 mg mMT-Ⅰ;원자흡수측정표명순화득도적mMT-Ⅰ적력리자결합능력접근우천연MT.
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, metalbinding activity of the purified mMT- Ⅰ was almost the same as that of wild type MT.