PMC:103662 / 26932-28741 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/103662","sourcedb":"PMC","sourceid":"103662","source_url":"https://www.ncbi.nlm.nih.gov/pmc/103662","text":"Protein expression and detergent extraction\nProteorhodopsin was expressed by E. coli strain UT5600 containing an additional plasmid encoding for the first-reported pR gene (accession #AF279106, obtained from the uncultured proteobacterium EBAC31A08 clone BAC [1]) with an Ara promoter and ampicillin resistance (kindly provided by O. Béjà). Single colonies were selected and grown overnight in LB/amp media (200 ml, 37°C, 300 rpm). This culture was then diluted 10× into several 500-ml cultures. After a further 2 h incubation in the shaker bath, a stock solution of 20% L-arabinose was added, to give a final concentration of 0.2% L-arabinose. This culture was then incubated for 4 h (37°C, 300 rpm). The cells (~20 ml wet volume) were then collected by centrifugation (6000 rpm × 30 min) and washed 3× with 100 mM HEPES, pH 7.1 (buffer A). The cells were then resuspended in buffer A and incubated at 4°C with 50 μg all-trans-retinal (added as a concentrated ethanol solution) for 3 h. The cells were collected by centrifugation (6000 rpm × 30 min), then resuspended in 60 mL buffer A containing 0.3 mg/mL lysozyme, and stirred for 4 h at room temperature. The cells were again collected by centrifugation (6000 rpm × 30 min), then lysed with 20 ml of 20% sodium cholate, pH 7.1 (30 min., 4°C). The cells were centrifuged again (6000 rpm × 30 min), and the supernatants collected. After extracting 3× more with the same cholate solution, the pooled supernatants were diluted 10× with buffer A and centrifuged at 180,000 g for 45 min to collect the membrane pellet. This cholate-washed membrane pellet was then further extracted 3× with 3.0% β-octyl-D-glucoside (OG) in buffer A (30 min, with stirring, 4°C). The pooled supernatants, containing OG-solubilized pR (~15 mg), were then diluted 6× with buffer A.","divisions":[{"label":"title","span":{"begin":0,"end":43}}],"tracks":[]}