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.