利用内蛋白子剪切功能一步纯化重组人神经营养因子-3  

One Step Purification for Recombinant Human Neurotrophic Factor-3 with the Splicing Function of Intein

作　　者：袁静明[1] 李卓玉[1] 王雅梅[1] 徐明群[2] sxu.edu.cn 

机构地区：[1]山西大学生物工程中心,太原030006 [2]NewEnglandBiolabs,Beverly

出　　处：《中国生物化学与分子生物学报》2000年第3期335-339,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金! (3 95 70 1 5 4 );山西省自然科学基金项目

摘　　要：将人神经营养因子 - 3(h NT3)基因插入含内蛋白子 -几丁质结合区 (Intein- CBD)片段的质粒p TXB1的多克隆位点 ,构建成重组子 p TXB- h NT3,随后转化入 E.coli 2 566并进行融合表达 .表达产物包涵体经 8mol/ L脲变性 ,并在 GSH,GSSG存在下复性 .复性后的融合蛋白经几丁质珠亲和柱吸附 .待洗涤杂蛋白后 ,加入 50 mmol/ L DTT在 4℃或 2 5℃进行剪切反应 48h,再用缓冲液洗脱 ,即得 h NT3.SDS- PAGE分析表明 ,h NT3达电泳纯 .其分子量约为 1 4 kHuman neurotrophic factor 3(hNT3) was amplified from human whole blood DNA by PCR and it was inserted into the multi cloning sites of a special vector pTXB1, which contained the DNA fragment coding for intein chitin binding domain (intein CBD) to construct the recombinant plasmid pTXB hNT3. After transferring plasmid pTXB hNT3 into the host E.coli 2566, the strain was cultured in LB medium and induced by IPTG. The fusion protein, hNT3 Intein CBD,was mainly expressed as the form of inclusion bodies. In order to make it soluble, the inclusion bodies were denatured in 8 mol/L urea and renatured in the presence of 1.7 mol/L urea containing GSH, GSSG and L arginine. The soluble product possibly with its correct conformation was slowly loaded on a chitin beads column which was pre equilibrated with 20 mmol/L Hepes buffer, pH 8.0, including 1.7 mol/L urea and then was washed with the same buffer to harbor the fusion protein and remove the other proteins. After that, a solution of 50 mmol/L DTT in the same buffer was rapidly through the column and the cleavage reaction was carried out under intein splicing conditions for 48 h at 4℃ or 25℃. Finally, hNT3 was eluated from the column with the washing buffer and examined on SDS PAGE. The results showed that hNT3 intein CBD could be partially cleaved by DTT and hNT3 was purified through one step procedure on chitin beads column.

关 键 词：人神经营养因子-3 内蛋白子 DTT剪切 纯化 

分 类 号：R971[医药卫生—药品] TQ93[医药卫生—药学]