›› 2012, Vol. 32 ›› Issue (5): 580-.doi: 10.3969/j.issn.1674-8115.2012.05.010

• Original article (Basic research) • Previous Articles     Next Articles

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

ZHANG Zhen-zhen1, YIN Yan-qing2, ZHOU Jia-wei2, LE Wei-dong1   

  1. 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:2012-05-28 Published:2012-06-01

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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.

Key words: p43/AIMP1, protein expression and purification, GST pull-down, neurofilament light subunit