GSK3β及其显性负突变体的原核表达与纯化
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国家自然科学青年科学基金资助(21310037)


Prokaryotic expression and purification of glycogen synthase kinase 3β and its dominant negative mutant
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    摘要:

    目的:构建原核表达GST-GSK3β1-GST-GSK3β2及其显性负突变体(dominant-negative mutant)GST-GSK3β1 K85M的重组质粒,并进行融合蛋白的表达-纯化及鉴定-方法:利用PCR扩增和基因重组技术,以含有人源目的片段的质粒Flag-GSK3β1-YFP-GSK3β2及Flag-GSK3β1 K85M为模板,扩增出相应的目的基因片段,并将其插入具有GST标签的原核表达载体pGEX-6P-1中,构建GST融合蛋白表达质粒-通过对GST-GSK3β1-GST-GSK3β2和GST-GSK3β1 K85M融合蛋白进行诱导表达-温和法裂解以及纯化实验,确立有效的表达和纯化方法-最后应用Western blot方法鉴定纯化的融合蛋白-结果:成功构建GST-GSK3β及其显性负突变体质粒,并通过诱导表达和纯化得到目的蛋白-结论:GST-GSK3β1-GST-GSK3β2及GST-GSK3β1 K85M显性负突变体质粒的成功构建及目的蛋白的表达与纯化,为更深入地研究GSK3β蛋白的生物学功能奠定基础-

    Abstract:

    Objective: To construct prokaryotic expression recombination plasmids containing GST-GSK3β1,GST-GSK3β2 and dominant-negative mutant GST-GSK3β1 K85M,then express,purify and identify these three fusion proteins. Methods: Using PCR and recombinant DNA technology,the target genes were amplified from templates Flag-GSK3β1,YFP-GSK3β2 and Flag-GSK3β1 K85M respectively and inserted into the prokaryotic expression vector pGEX-6P-1 with glutathione-S-transferase(GST) tag to construct the expression plasmids expressing GST-GSK3β1,GST-GSK3β2 and GST-GSK3β1 K85M fusion proteins. An effective mean of expression and purification was established by a series of expression,gentle lysis and purification experiments. In the end,Western blot was used to identify the purified fusion protein. Results: We succeed in constructing wild type and dominant-negative mutant plasmids of GST-GSK3β,expressing and purifying these fusion proteins. Conclusion: The successful construction,expression and purification of those target proteins facilitate us to further study the biology function of GSK3β.

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王建瑛,高玲梅,刘小会,高学娟,刘朗夏. GSK3β及其显性负突变体的原核表达与纯化[J].南京医科大学学报(自然科学版),2011,(11):1556-1560

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  • 收稿日期:2011-07-05
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