| Karpushova A, Brümmer F, Barth S, Lange S, Schmid RD.
(2005)
Appl microbiol biotechnol,
67,
59-69 |
| Purification & characterization |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 30.0 |
| 4 h |
| pET-estB2 |
| E. coli BL21(DE3) cells were freshly transformed with the expression vectors pET-estB1 and pET-estB2 and grown at 37°C in 400 ml LB media until the early exponential phase (OD600=0.4–0.8). EstB1 and EstB2 production was induced with 0.01–1 mM IPTG, and cultivation was continued at 30°C for 4 h. E. coli cells were then harvested by centrifugation (4,000 g) and washed twice with 50 mM potassium phosphate buffer, pH 7.5, 4°C. E. coli cell extracts were prepared by sonication [3 times for 2 min, the power level set between 4 and 5, 50% output, Branson Sonifier 250 (Branson, Dietzenbach, Germany)] and used for purification or to assay esterase activity. |
| IPTG |
| OD n/a =
n/a |
| Sonication |
| None |
| None |
| insoluble |
| Dialysis |
| n/a |
| 50 mM potassium phosphate buffer, pH 7.5, 5 M urea, 1 mM DTT and 1 mM EDTA |
| 50 mM potassium phosphate buffer, pH 7.5, 1 mM DTT and 1 mM EDTA |
| None |
| no |
| 7.5 |
| 4.0 |
| n/a |
| 10-12 h |
| DTT |
| 1 mM |
| Refolding of EstB2
Isolation of inclusion bodies was performed according to the method of Rudolph and Lilie (Rudolph and Lilie 1996). The pure inclusion bodies were dissolved in extraction buffer (50 mM potassium phosphate buffer, pH 7.5, 5 M urea, 1 mM DTT and 1 mM EDTA) to a final protein concentration of 1 mg/ml and incubated for 3 h at room temperature (RT), with slight agitation. The solubilised inclusion bodies were dialysed at 4°C for 10–12 h with 50 mM potassium phosphate buffer, pH 7.5, 1 mM DTT and 1 mM EDTA. |
| SDS-PAGE,Activity assay |
| None |
| None |
| n/a |
| n/a |
| n/a |
| n/a |