Cloning, overexpression, purification, and characterization of receptor-interacting protein 3 truncation in Escherichia coli.

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 in Escherichia 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 alpha-helix structure and temperature-dependent conformational change.

authors

Jeong MS,Park JS,Jang SB

doi

10.1007/BF02729060

subject

Has Abstract

pub_date

2007-05-01 00:00:00

pages

175-86

issue

2-3

eissn

0273-2289

issn

1559-0291

pii

ABAB:141:2-3:175

journal_volume

141

pub_type

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