Cloning, overexpression, purification, and characterization of receptor-interacting protein 3 truncation in Escherichia coli.
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.
Jeong
Mi Suk
MS
Department of Molecular Biology, Pusan National University, Jangjeondong, Keumjeong-gu, Busan, 609-735, Korea.
Park
Jeong Soon
JS
Jang
Se Bok
SB
D000595:Amino Acid Sequence; D000838:Anions; D002852:Chromatography, Ion Exchange / Q000379:methods; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics*; D013696:Temperature
D000838:Anions; D017931:DNA Primers; D005982:Glutathione Transferase; C119705:RIPK3 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases