| Booth, C., Pulaski, L., Gottesman, M. M., Pastan, I.
(2000)
Biochemistry,
39,
5518-5526 |
| Structure-Function |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(lambdaDE3) |
| 37.0 |
| 90min |
| pET3a |
| Cells were grown overnight on LB agar containing 100micgrog/ml ampicillin and superbroth (2% glucose, 0.05% MgSO4). After induction cells were harvested via centrifugation(3500rpm, 30min) Pellet was washed by sequential resuspension and centrifugation(13,000rpm, 50min) in 200ml of buffer A(50mM Tris and 20mM EDTA, pH 8.0). During initial washes buffer A contained 2.5% Triton X-600, this was then decreased to 1%, prior to three washes in the absence of detergent. The final pellet was resuspended in 20ml of buffer A and then centrifuged(20,000rpm, 20min) to obtain inclusion bodies. |
| IPTG |
| OD 600 =
3 |
| Not stated |
| Detergents |
| None |
| insoluble |
| Dilution/Dialysis combination |
| n/a |
| 6M GuHCl, 0.1M Tris, 2mM EDTA, pH 8.0 |
| 0.1M Tris, 0.5M arginine, 2mM EDTA, pH 8.0 |
| None |
| no tag |
| 8.0 |
| 10.0 |
| n/a |
| n/a |
| None |
| n/a |
| Inclusion body were solubilized and centrifuged(20,000rpm, 20min) and stored at room temp for at least 2 hours. Solubilized inclusion bodies were harvested by centrifugation(20,000rpm, 30min) before renaturation. Refolding was preformed by a rapid 100 fold dilution in refolding buffer, this was maintained at 10degC for 12hrs, before overnight dialysis against 20mM Tris, pH 8.0/ 100mM NaCl. |
| Far-UV Circular Dichroism,Enzyme activity,SDS-PAGE |
| None |
| L-Arginine,Glycerol,Triton X-100 |
| n/a |
| n/a |
| n/a |
| n/a |