PMC:4528928 / 3633-5274 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4528928","sourcedb":"PMC","sourceid":"4528928","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4528928","text":"2.1. Protein expression and purification   \nThe sequence encoding the N-terminal domain of hMLH1 (residues 1–340) was amplified by PCR and subcloned into the pET-28-MHL vector (GenBank deposition ID EF456735) downstream of the polyhistidine affinity tag. The protein was overexpressed in Escherichia coli BL21 (DE3) V2R-pRARE cells in Terrific Broth medium in the presence of 50 µg ml−1 kanamycin. The cells were grown at 37°C to an OD600 nm of 1.5, induced by the addition of 1 mM isopropyl β-d-1-thio­galactopyranoside (IPTG) and incubated overnight at 15°C. The cells were harvested by centrifugation at 7000 rev min−1 and resuspended in 50 mM HEPES pH 7.4, 500 mM NaCl, 2 mM β-mercaptoethanol, 5% glycerol, 0.1% CHAPS, 1 mM phenylmethylsulfonyl fluoride (PMSF). The cells were lysed by passage through a microfluidizer (Microfluidics Corporation) at 138 MPa. After clarification of the crude extract by high-speed centrifugation, the lysate was applied onto a 5 ml HiTrap Chelating column (GE Healthcare) charged with Ni2+. The column was washed with ten column volumes of 20 mM HEPES pH 7.4 containing 500 mM NaCl, 50 mM imidazole and 5% glycerol. The protein was eluted in 20 mM HEPES pH 7.4, 500 mM NaCl, 250 mM imidazole, 5% glycerol and then loaded onto a Superdex 200 (26/60, GE Healthcare) column equilibrated in 20 mM PIPES pH 6.5 buffer containing 250 mM NaCl. TEV protease was added to the combined fractions containing MLH1. The protein was further purified to homogeneity by ion-exchange chromatography on a Source 30S column (10/10; GE Healthcare) and eluted in a final buffer consisting of 20 mM PIPES pH 6.5, 250 mM NaCl.","divisions":[{"label":"Title","span":{"begin":6,"end":44}}],"tracks":[]}