Expression, purification, and characterization of the E1 for human NEDD8, the heterodimeric APPBP1-UBA3 complex.
The NEDD8 pathway is important for numerous biological processes, including cell proliferation, signal transduction, and development. The heterodimeric activating enzyme of NEDD8, APPBP1-UBA3, plays an essential role in NEDD8 conjugation. Not surprisingly, mutations in APPBP1 and UBA3 lead to defects in many cellular functions. The APPBP1-UBA3 complex initiates NEDD8 conjugation by first catalyzing adenylation of the C terminus of NEDD8 and ultimately catalyzing transfer of NEDD8 to the downstream enzyme in the pathway, Ubc12. This chapter describes methods for expressing and purifying APPBP1-UBA3 for in vitro studies of NEDD8 conjugation.
Huang
Danny T
DT
Department of Structural Biology and Genetics/Tumor Cell Biology, St. Jude's Children's Research Hospital, Memphis, TN 38105, USA.
Schulman
Brenda A
BA
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D000075687:NEDD8 Protein; D044764:Ubiquitin-Activating Enzymes; D044763:Ubiquitin-Conjugating Enzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D014452:Ubiquitins / Q000737:chemistry / Q000378:metabolism*
D004268:DNA-Binding Proteins; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D014452:Ubiquitins; D044763:Ubiquitin-Conjugating Enzymes; D044764:Ubiquitin-Activating Enzymes; C491802:NAE protein, human