Recombinant Human NEDD8 can be conjugated to substrate proteins via the subsequent actions of a NEDD8-activating (E1) enzyme, a NEDD8-conjugating (E2) enzyme, and a NEDD8 ligase (E3). Reaction conditions will need to be optimized for each specific application. We recommend an initial Recombinant Human NEDD8 concentration of 10-50 μM.
>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain
8.6 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
Neural Precursor Cell Expressed Developmentally Downregulated Gene 8 (NEDD8), also known as Related to Ubiquitin 1 (Rub1), is a 6-8 kDa member of the Ubiquitin family of proteins. Human pro-NEDD8 is 81 amino acids (aa) in length. Pro-NEDD8 contains one Ubiquitin-like domain (aa 1-65) and a 4-5 aa C-terminal propeptide (1,2). Cleavage of the propeptide exposes a C-terminal glycine residue that is used to generate a glycine-lysine intermolecular bond. Mature human NEDD8 shows 100% aa identity to NEDD8 from mouse, rat, and canine. Human NEDD8 is activated by a distinct NEDD8-activating (E1) enzyme, a heterodimeric complex composed of APPBP1 and UBA3 subunits (3). Activated NEDD8 is subsequently transferred to the UBE2M/Ubc12 or UBE2F NEDD8-conjugating (E2) enzymes (4). Through a process termed neddylation, the ROC1/Rbx1 RING Finger E3 ligase transfers NEDD8 to specific substrates (5). For example, transfer of NEDD8 to the Cullin scaffold activates members of the Cullin-RING Ligase (CRL) family of Ubiquitin ligases (E3) (6-8). CRL-induced ubiquitination is known to regulate proteins that are important for cell cycle progression and cell survival (9,10). Physiologically, NEDD8 plays a critical regulatory role in cell proliferation and dysregulation of the NEDD8 pathway has been associated with several cancer pathologies (11,12).
1. Kumar, S. et al. (1993) Biochem. Biophys. Res. Commun. 195:393. 2. Kamitani, T. et al. (1997) J. Biol. Chem. 272:28557. 3. Huang, D.T. & B.A. Schulman (2005) Methods Enzymol. 398:9. 4. Gong, L. & E.T. Yeh (1999) J. Biol. Chem. 274:12036. 5. Ohki, Y. et al. (2009) Biochem. Biophys. Res. Commun. 381:443. 6. Petroski, M.D. & R.T. Deshares (2005) Nat. Rev. Mol. Cell Biol. 6:9. 7. Hjerpe, R. et al. (2012) Biochem. J. 441:927. 8. Calabrese, M.F. et al. (2011) Nat. Struct. Mol. Biol. 18:947. 9. Tateishi, K. et al. (2001) J. Cell. Biol. 155:571. 10. Ohh, M. et al. (2002) EMBO Rep. 3:177. 11. Luo, Z. et al. (2012) Cancer Res. 72:3360. 12. Soucy, T.A. et al. (2010) Genes Cancer 1:708.
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