论著(基础研究)

GST-p43/AIMP1融合蛋白表达和纯化以及与NF-L的相互作用

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  • 1.上海交通大学 医学院附属瑞金医院神经科, 上海 200025; 2.中国科学院上海生命科学研究院 |神经科学研究所, 上海 200031
张珍珍(1984—), 女, 硕士生;电子信箱: zhen-0720@163.com。

网络出版日期: 2012-06-01

Expression and purification of GST-p43/AIMP1 fusion protein and its interaction with NF-L

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  • 1.Department of Neurology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China;2.Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

Online published: 2012-06-01

摘要

目的 构建人p43/AIMP1蛋白与谷胱甘肽转移酶(GST)重组的融合蛋白原核表达载体并进行表达纯化,通过GST pull-down方法验证其与神经中间丝轻链(NF-L)的体外直接相互作用。方法 以重组质粒pcDNA3.1-p43为模板,扩增p43/AIMP1基因,产物经纯化回收后与原核表达载体pGEX4T-1连接构建成新载体GST-p43/AIMP1,经鉴定完全正确后转化大肠埃希菌BL21(DE3),通过异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导表达,并纯化获得目的蛋白;将myc-NF-L体外转染HEK293T细胞,利用GST pull-down原理和方法验证人p43蛋白与神经中间丝轻链蛋白NF-L之间的相互作用。结果 酶切鉴定和测序结果显示,成功构建了GST-p43/AIMP1融合蛋白原核表达载体;考马斯亮蓝染色和Western blotting结果显示,成功获得有生物活性的GST-p43/AIMP1融合蛋白;GST pull-down实验结果证实,p43/AIMP1与NF-L存在直接相互作用。结论 获得有生物活性的GST-p43/AIMP1蛋白,并成功应用GST pull-down方法证实p43/AIMP1与NF-L在体外存在直接的相互作用。

本文引用格式

张珍珍, 尹延青, 周嘉伟, 等 . GST-p43/AIMP1融合蛋白表达和纯化以及与NF-L的相互作用[J]. 上海交通大学学报(医学版), 2012 , 32(5) : 580 . DOI: 10.3969/j.issn.1674-8115.2012.05.010

Abstract

Objective To construct and purify the prokaryotic expression vector of fusion protein of human p43/AIMP1 protein and glutathione-S-transferase (GST), and verify its direct interaction with neurofilament light subunit (NF-L) in vitro through GST pull-down assay. Methods p43/AIMP1 gene was amplified from pcDNA3.1-p43, and was inserted into prokaryotic expression vector pGEX4T-1 to generate novel vector GST-p43/AIMP1. After identification of GST-p43/AIMP1, Escherichia coli BL21 (DE3) was transfected, which was induced by isopropyl-β-D-thiogalactoside (IPTG), and target protein was obtained after purification. HEK293T cells were transfected in vitro with myc-NF-L, and the interaction between GST-p43/AIMP1 and myc-NF-L was detected using GST pull-down assay. Results Restriction enzyme digestion and sequencing indicated that prokaryotic expression vector of GST-p43/AIMP1 fusion protein was successfully constructed. Coomassie brilliant blue staining and Western blotting revealed that GST-p43/AIMP1 fusion protein with bioactivity was successfully obtained. GST pull-down assay verified that there was direct interaction between p43/AIMP1 and NF-L. Conclusion The fusion protein of GST-p43/AIMP1 with bioactivity has been successfully obtained, and the direct interaction between p43/AIMP1 and NF-L has been verified in vitro through GST pull-down assay.

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