PMC:101403 / 19051-20481 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/101403","sourcedb":"PMC","sourceid":"101403","source_url":"https://www.ncbi.nlm.nih.gov/pmc/101403","text":"E. coli strain BL21(DE3) was transformed with pETY87G2A.14. A single colony was picked from an LB agar plate containing 50 μg/ml ampicillin and inoculated into 10 ml LB medium containing 50 μg/ml ampicillin and incubated at 37°C. When the cells reached an A600 of 0.5, they were transferred to 1 litre of fresh LB medium containing 50 μg/ml ampicillin and grown to an A600 of 0.3 at 37°C, then transferred to an incubator at 25°C. Isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added to 1 mM at an A600 of 0.8, and the cells induced for 8 h. The induced cells (4 g) were harvested, washed and resuspended in 20 ml breakage buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM DTT). The cell suspension was sonicated and the cell lysate was then cleared by centrifugation at 10,000 × g at 4°C for 15 min. The supernatant was applied to a 15 × 50 mm column of NiCAM™-HC resin (Sigma) equilibrated with 50 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 1 mM 2-mercaptoethanol at a flow rate of 0.5 ml/min. After eluting the unbound proteins, a linear gradient of 0–40 mM histidine in the same equilibration buffer was applied at flow rate of 1 ml/min and 1 ml fractions collected and analysed by SDS-PAGE. Those containing pure Trx-Y87G2A.14 fusion protein were collected, dialysed overnight at 4°C against 1 litre of 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, ImM DTT and then concentrated by ultraflltration (Amicon) and stored at -20°C in 50% glycerol.","tracks":[]}