Development of an efficient E. coli expression and purification system for a catalytically active, human Cullin3-RINGBox1 protein complex and elucidation of its quaternary structure with Keap1.
The Cullin3-based E3 ubiquitin ligase complex is thought to play an important role in the cellular response to oxidative stress and xenobiotic assault. While limited biochemical studies of the ligase's role in these complex signaling pathways are beginning to emerge, structural studies are lagging far behind due to the inability to acquire sufficient quantities of full-length, highly pure and active Cullin3. Here we describe the design and construction of an optimized expression and purification system for the full-length, human Cullin3-RINGBox 1 (Rbx1) protein complex from Escherichia coli. The dual-expression system is comprised of codon-optimized Cullin3 and Rbx1 genes co-expressed from a single pET-Duet-1 plasmid. Rapid purification of the Cullin3-Rbx1 complex is achieved in two steps via an affinity column followed by size-exclusion chromatography. Approximately 15mg of highly pure and active Cullin3-Rbx1 protein from 1L of E. coli culture can be achieved. Analysis of the quaternary structure of the Cullin3-Rbx1 and Cullin3-Rbx1-Keap1 complexes by size-exclusion chromatography and analytical ultracentrifugation indicates a 1:1 stoichiometry for the Cullin3-Rbx1 complex (MW=111kDa), and a 1:1:2 stoichiometry for the Cullin3-Rbx1-Keap1 complex (MW=280kDa). This latter complex has a novel quaternary structural organization for cullin E3 ligases, and it is fully active based on an in vitro Cullin3-Rbx1-Keap1-Nrf2 ubiquitination activity assay that was developed and optimized in this study.
Small
Evan
E
Department of Biochemistry, University of Illinois at Chicago, Chicago, IL 60607, USA.
Eggler
Aimee
A
Mesecar
Andrew D
AD
D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D002384:Catalysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D044842:Cullin Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry*; D000072019:Kelch-Like ECH-Associated Protein 1; D020836:Protein Structure, Quaternary; D054875:Ubiquitination; D014461:Ultracentrifugation
C503645:CUL3 protein, human; D002352:Carrier Proteins; D044842:Cullin Proteins; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; C479529:RBX1 protein, human