Recombinant Human O-6-methylguanine-DNA Methyltransferase, His-tagged

• Cat.No.: MGMT-121H
• Product Overview: Recombinant MGMT, fused to His-tag at N-terminus, was expressed in E.coli and was purified by conventional chromatography techniques,23.8 kDa (227 aa), (Real molecular weight on SDS-PAGE will be shift up).

Specification

• Species: Human
• Source: E.coli
• Tag: His
• Description: O-6-methylguanine-DNA methyltransferase (MGMT) is an enzyme that repairs O-6-methylguanine, a mutagenic DNA base damaged by endogenous and environmental alkylating agents and is involved in the cellular defense against the biological effects of O-6-methylguanine in DNA. MGMT repairs alkylated guanine in DNA by stoichiometrically transferring the alkyl group at the O-6 position to a cysteine residue in the enzyme. There are few reports that abnormal MGMT expression correlates with the prognosis in human solid cancers.
• Sequences of amino acids: MGSSHHHHHH SSGLVPRGSH MDKDCEMKRT TLDSPLGKLE LSGCEQGLHE IKLLGKGTSA ADAVEVPAPA AVLGGPEPLM QCTAWLNAYF QPEAIEEFP VPAFHHPVFQ QESFTRQVLW KLLKVVKFGE VISYQQLAAL AGNPKAARAV GGAMRGNPVP ILIPCHRVVC SSGAVGNYSG GLAVKEWLLA HEGHRLGKPG LGGSSGLAGA WLKGAGATSG SPPAGRN
• Formulation: Liquid. In 20 mM Tris-HCl buffer (pH 7.5) containing 1 mM DTT, 10% glycerol
• Purity: > 95% by SDS - PAGE
• Concentration: 0.5 mg/ml (determined by Bradford assay)
• Endotoxin Level: < 1.0 EU per 1 g of protein (determined by LAL method)
• Storage: Can be stored at +4°C short term (1-2 weeks). For long term storage, aliquot and store at -20°C or -70°C. Avoid repeated freezing and thawing cycles.
• Publications:
  Fluorogenic Real-Time Reporters of DNA Repair by MGMT, a Clinical Predictor of Antitumor Drug Response (2016)
  Activatable Probes for Anti-Cancer Therapy (2019)

Gene Information

• Gene Name: MGMT O-6-methylguanine-DNA methyltransferase [ Homo sapiens ]
• Synonyms: O-6-methylguanine-DNA-alkyltransferase; O-6-methylguanine-DNA methyltransferase; EC 2.1.1.63; 6-O-methylguanine-DNA methyltransferase; OTTHUMP00000020741; MGMT; O6-methylguanine-DNA methyltransferase; methylguanine-DNA methyltransferase
• Gene ID: 4255
• mRNA Refseq: NM_002412
• Protein Refseq: NP_002403
• MIM: 156569
• UniProt ID: P16455
• Chromosome Location: 10q26
• Pathway: DNA Repair
• Function: DNA binding; DNA-methyltransferase activity; metal ion binding; transferase activity ; methylated-DNA-[protein]-cysteine S-methyltransferase activity; zinc ion binding ; methylated-DNA-[protein]-cysteine S-methyltransferase activity.

Citation

Fluorogenic Real-Time Reporters of DNA Repair by MGMT, a Clinical Predictor of Antitumor Drug Response

Journal: PLoS ONE    PubMed ID: 27035132    Data: 2016/4/1

Authors: Andrew A. Beharry, Zachary D. Nagel, Robert W Sobol

Article Snippet:Purified recombinant human MGMT (His-tagged, expressed in E . Coli ) was purchased from Creative BioMart.. All assays were carried out at 37°C in 70 mM HEPES buffer (pH 7.8) containing 5 mM EDTA, 1 mM dithiothreitol and 50 μg/ml BSA.All assays were carried out at 37°C in 70 mM HEPES buffer (pH 7.8) containing 5 mM EDTA, 1 mM dithiothreitol and 50 μg/ml BSA.

Fluorescence spectra showing the overall fold-changes in intensity observed when comparing fluorescence measured before (dashed line) and after (solid line) addition of purified MGMT protein are shown at left of each figure. Time courses (on the right) show time-dependent fluorescence increases immediately after addition of enzyme. Final probe and MGMT concentrations were 100 nM. Assays were run at 37°C in 70 mM HEPES buffer pH 7.8 containing 5 mM EDTA, 1 mM dithiothreitol and 50 μg/ml BSA. (A) chemosensor 1 containing dT FAM , (B), chemosensor 2 containing Cy3, (C), chemosensor 3 containing dT TMR and (D), chemosensor 4 containing perylene nucleoside. Measurements were repeated 3 times. Standard deviations are provided in .

Incubation of purified MGMT enzyme with the inhibitors BG and PaTrin-2 led to a concentration dependent decrease in observed final fluorescence intensity, indicative of MGMT inhibition. MGMT (10 nM) was incubated with inhibitor for 10 min at 37°C in 70 mM HEPES buffer (pH 7.8) containing 5 mM EDTA, 1 mM dithiothreitol and 50 μg/ml BSA. Final fluorescence was acquired 10 min after addition of probe (10 nM). Data were normalized to measurements without inhibitor. Each data point is the average of 3 measurements.