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Cloning, overexpression, purification, and characterization of receptor-interacting protein 3 truncation inEscherichia coli
Authors:Mi Suk Jeong  Jeong Soon Park  Se Bok Jang
Affiliation:(1) Department of Molecular Biology, Pusan National University, Jangjeondong, Keumjeong-gu, 609-735 Busan, Korea;(2) Present address: Research Center for Advanced Science and Technology, Dongseo University, 617-716 Busan, Korea
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.
Keywords:Subcloning  overexpression  purification  characterization  receptor-interacting protein 3
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