Original article (Basic research)

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

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.

Cite this article

ZHANG Zhen-zhen, YIN Yan-qing, ZHOU Jia-wei, et al . Expression and purification of GST-p43/AIMP1 fusion protein and its interaction with NF-L[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2012 , 32(5) : 580 . DOI: 10.3969/j.issn.1674-8115.2012.05.010

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