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Cloning, overexpression, purification, and characterization of receptor-interacting protein 3 truncation inEscherichia coli

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Abstract

To facilitate structural studies of receptor-interacting protein 3 (RIP3), we developed a large-scale expression system of a glutathione-S-transferase (GST) fused with an 82 amino acid RIP3 protein inEscherichia coli. RIP3 truncation was subcloned into the pGEX-4T-1 vector and overexpressed in BL21(DE3)RIL cells. The soluble RIP3 protein was successfully purified to homogeneity using GST tag, an anion-exchange column, and gel filtration chromatography. The purity, identity, and conformation of the RIP3 protein were determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, matrix-assisted laser desorption ionization mass spectrometry, circular dichroism, and fluorescence spectroscopic studies. RIP3 showed dominance of the α-helix structure and temperature-dependent conformational change.

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Author notes

  1. Mi Suk Jeong

    Present address: Research Center for Advanced Science and Technology, Dongseo University, 617-716, Busan, Korea

Authors and Affiliations

  1. Department of Molecular Biology, Pusan National University, Jangjeondong, Keumjeong-gu, 609-735, Busan, Korea

    Mi Suk Jeong, Jeong Soon Park & Se Bok Jang

Authors
  1. Mi Suk Jeong
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  2. Jeong Soon Park
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  3. Se Bok Jang
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Correspondence to Se Bok Jang.

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Jeong, M.S., Park, J.S. & Jang, S.B. Cloning, overexpression, purification, and characterization of receptor-interacting protein 3 truncation inEscherichia coli . Appl Biochem Biotechnol 141, 175–186 (2007). https://doi.org/10.1007/BF02729060

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  • DOI: https://doi.org/10.1007/BF02729060

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