SAE1 [GST-tagged] / SAE2 [6His-tagged]


Catalogue Number
61-0005-050
Product Size
50 µg
Price £
£325
Accession Number
NP_005491.1 / NP_005490.1
Residues Expressed
1-346 / 1-640
Alternate Product Size
10 µg
Certificate of Analysis Size
50 pg
Species
Human
Source
E.coli
Quantity
50 µg
Storage
-70°C
Concentration
0.5 mg/ml
Formulation
50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
Molecular Weight
SAE1 = 64.8 kDa SAE2 = 73.4 kDa
Stability
12 months at -70°C; aliquot as required
Protein Sequence
Accession number: SAE1 = NP_005491.1 and SAE2 = NP_005490.1 For full protein sequence information download the Certificate of Analysis pdf.
QA; Protein Identification
Confirmed by mass spectrometry.
QA Activity

E1 Thioester SUMO Loading Assay: The activity of GST-SAE1/His-SAE2 was validated by loading SUMO1 onto the active cysteine of GST-SAE1/His-SAE2. Incubation of the GST-SAE1/His-SAE2 enzyme in the presence of SUMO1 and ATP at 30°C was compared at two time points, T0 and T10 minutes. Sensitivity of the SUMO / GST-SAE1/His-SAE2 thioester bond to the reducing agent DTT was confirmed.


Background

The enzymes of the SUMOylation pathway play a pivotal role in a number of cellular processes including nuclear transport, signal transduction, stress responses and cell cycle progression. The covalent modification of proteins by small ubiquitin-related modifiers (SUMOs) may modulate their stability and subcellular compartmentalisation. Three classes of enzymes are involved in the process of SUMOylation; an activating enzyme (E1), conjugating enzyme (E2) and protein ligases (E3s). SAE1/SAE2 is a SUMO1, 2 and 3 E1 activating enzyme and functions as a heterodimer. Cloning of the human SAE1 and SAE2 genes was first described by Desterro et al. (1999). SAE1 and SAE2 share sequence similarity to the N-terminus and C-terminus of ubiquitin E1 activating enzymes respectively (Desterro et al. 1999). SAE2 harbours the E1-like active cysteine site while SUMO1 transfer to the E2 conjugating enzyme UBE2I requires both of the SAE subunits (Desterro et al. 1999). A crystal structure of the SAE1/SAE2 dimer together with the SUMO1 adenylate has been solved at 2.45 Ångström resolution (Olsen et al. 2010). Western blot analysis of cell-cycle synchronised HeLa cells demonstrated increased SAE1 expression in S phase followed by a decrease in G2 phase. Immunofluorescence showed that SAE1 and SAE2 were distributed throughout the nuclei but were excluded from the nucleoli (Azuma et al. 2001). A short hairpin RNA (shRNA) screen was carried out in the presence of aberrant MYC signalling to identify genes that altered the fitness of mammary epithelial cells. In this screen SAE1 and SAE2 were identified as MYC synthetic lethal genes. Upon MYC hyperactivation inactivation of SAE2 led to mitotic catastrophe and cell death and it is thought that SAE2 inactivation could be a therapeutic strategy in MYC driven cancers (Kessler et al. 2012).


References

Azuma Y, Tan SH, Cavenagh MM, Ainsztein AM, Saitoh H, et al. (2001) Expression and regulation of the mammalian SUMO-1 E1 enzyme. FASEB J 15, 1825-1827.

Desterro JM, Rodriguez MS, Kemp GD, Hay RT (1999) Identification of the enzyme required for activation of the small ubiquitin-like protein SUMO-1. J Biol Chem 274, 10618-10624.

Kessler JD, Kahle KT, Sun T, Meerbrey KL, Schlabach MR, et al. (2012) A SUMOylation-dependent transcriptional subprogram is required for Myc-driven tumorigenesis. Science 335, 348-353.