High-level expression and purification of recombinant SCF ubiquitin ligases.
The SCF complexes are the prototype of a superfamily of cullin-dependent ubiquitin ligases, which regulate diverse cellular functions by promoting the ubiquitination of a large number of regulatory and signaling proteins. The SCF complexes are organized by the elongated scaffold protein subunit Cul1, which interacts with the Rbx1 RING finger protein at one end and the Skp1 adaptor protein at the other. By binding to Skp1, members of the F-box protein family are responsible for recruiting specific substrates to the ligase machine. This chapter describes methods that we have developed to achieve high-level expression and purification of two recombinant SCF complexes from both insect cells and bacteria. We emphasize the power of protein coexpression and a novel "Split-n-Coexpress" method in producing soluble and functional recombinant proteins and protein complexes. We propose that similar approaches can be used to obtain large quantities of other SCF and SCF-like complexes for biochemical and structural investigations.
Li
Ti
T
Department of Pharmacology, University of Washington, Seattle, WA 98195, USA.
Pavletich
Nikola P
NP
Schulman
Brenda A
BA
Zheng
Ning
N
D000818:Animals; D016367:Baculoviridae; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular / Q000379:methods*; D044842:Cullin Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D051379:Mice; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D044843:SKP Cullin F-Box Protein Ligases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D018411:Spodoptera / Q000235:genetics
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C100318:Cullin 1; D044842:Cullin Proteins; C479529:RBX1 protein, human; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D044843:SKP Cullin F-Box Protein Ligases