TDG SUMO1 was produced by co selleck chem inhibitor transforming the BL21 strain carrying the pGEX 6P 1 hTDG plas mid with the pT E1 E2 SUMO1 vector. Selection of BL21 colonies carrying both plasmids was performed by ampicilline chloramphenicol double selection as described. Unlabeled TDG SUMO1 was produced in LB medium and 15N labeled TDG SUMO1 in M9 minimal medium as previously described for TDG with 2. 5 g 15N labeled ammonium chloride as nitrogen source. The induction phase was performed overnight at 25 C with 0. 2 mM IPTG. The purification was realized as described for TDG with an additional intermediary purification step of cation exchange chromatography on HiTrap SP column. The column was equilibrated in 50 mM NaiPO4 pH 7.
4, 10% glycerol, 1 mM DTT containing 10 mM NaCl and TDG SUMO 1 protein was eluted at a flow rate of 2 mL min with a linear gra dient of NaCl from 0 to 100% buffer B in 5 column volumes. TDG mutants were expressed and purified following the same procedure as the wild type TDG protein. Expression profiles were comparable Inhibitors,Modulators,Libraries to wild type pro tein, but the protein quantities obtained for TDG D133A and TDG D133A E310Q after the first purifica tion step Inhibitors,Modulators,Libraries were significantly lower than for TDG wild type and TDG E310Q proteins. Protein protein interactions between TDG, TDG E310Q or SUMO conjugated TDG and SUMO 1 monitored by NMR spectroscopy NMR experiments were performed at 293 K on a Bruker DMX 600 MHz spectrometer equipped with a cryogenic triple resonance probe head. All 1H spectra were calibrated with 1 mM sodium 3 trimethylsilyl d propionate as a reference.
Batimastat All 1 fer composed of, 100 mM NaiPO4 pH 6. 6, 1 mM EDTA, 1 mM DTT, 5% D2O. 1H 15N HSQC Inhibitors,Modulators,Libraries spectra were recorded on 20 uM samples of 15N labeled proteins with at least 256 scans per increment and 128 dummy scans, 128 points in the nitrogen Inhibitors,Modulators,Libraries dimension and 1024 points in the proton dimension. Direct binding studies were performed by NMR spec troscopy on the 15N labeled isolated TDG N termi nus at 20 uM and a 3 fold excess of unlabeled SUMO 1, the 15N labeled TDG at 20 uM in presence of a 1, 3, 6, or 10 fold excess of unlabeled SUMO 1 and, conversely, 15N labeled SUMO 1 at 30 uM in presence of a 3 fold excess of unlabeled TDG or TDG E310Q. The 15N labeled TDG E310Q mutant and SUMO modified TDG was analyzed at 20 uM in presence of 10 equivalents SUMO 1.
Interactions of TDG, TDG N and SUMO 1 with G,T U containing dsDNA Annealing of oligonucleotides was performed by heating 1 mM solutions for 5 min at 100 C and cooling down the mixtures slowly to room temperature to obtain dou ble stranded 37 mers containing G,T or G,U mispairs. These etc solutions were lyophilized and dissolved at 50 uM final concentration in a 20 uM solution of 15N labeled TDG in a buffer constituted by 100 mM NaiPO4 pH 6. 6, 1 mM DTT and 1 mM EDTA. The SUMO 1 bind ing activity of TDG was investigated on a 20 uM solution of 15N TDG in presence of a 2.