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

Zhen Zhen Zhang, Yan Qing Yin, Jia Wei Zhou, Weidong Le

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)580-584
Number of pages5
JournalJournal of Shanghai Jiaotong University (Medical Science)
Volume32
Issue number5
DOIs
StatePublished - May 1 2012

Keywords

  • GST pull-down
  • Neurofilament light subunit
  • p43/AIMP1
  • Protein expression and purification

ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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