| Kati WM, Sham HL, McCall JO, Montgomery DA, Wang GT,
(1999)
Archives Biochemistry and Biophysics,
362,
363-375 |
| Undefined |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 3 h |
| pOM104 |
| For expression studies cultures were grown to an OD600 of 1.0 in “superbroth” (27), at which time protease 3C production was induced by the addition of either arabinose (to a final concentration of 0.2%) or IPTG (to 2 mM) as appropriate. Cell samples were collected at 0, 4, and 16 h postinduction. Large cultures of E. coli strain BL21(DE3)/pOM104, pOM62 were grown in 2800-ml Fernbach flasks containing 500 ml of medium per
flask which was supplemented with 200 ug/ml ampicillin and 15 ug/ml tetracycline. Cells were grown from a frozen seed to 0.6 – 0.8 OD600 at 37°C followed by induction with 100 mg/liter IPTG. Cells were grown for an additional 3 h and then harvested by centrifugation.
|
| IPTG |
| OD n/a =
n/a |
| French Press |
| None |
| not specified |
| insoluble |
| Dilution/Dialysis combination |
| 50 mM Tris–Cl, pH 7.5, containing 1 mM DTT, 1 mM MgCl 2, 10% sucrose, 50 ug/ml RNase, and 50 ug/ml DNase |
| 50 mM Tris–Cl, pH 7.9, containing 8 M urea, 1 mM EDTA, 1 mM DTT, 1 M NaCl, 0.5% NP-40, and 10% glycerol |
| 50 mM Tris–Cl, pH 7.9, 1 mM EDTA, 1 mM DTT, 1 M NaCl, 0.5% NP-40, and 10% glycerol |
| not specified |
| no tag |
| 7.9 |
| 0.0 |
| n/a |
| n/a |
| None |
| n/a |
| Purification and refolding of recombinant 3C protease. Cells were resuspended to a concentration of about 50 OD/ml in buffer A (50 mM Tris–Cl, pH 7.5, containing 1 mM DTT, 1 mM MgCl 2, 10% sucrose, 50 ug/ml RNase, and 50 ug/ml DNase). The cells were lysed by French press (American Instrument Company, Silver Spring, MD) and then the cell debris was pelleted by centrifugation in a Sorvall SA600 rotor at 4000 rpm for 10 min at 10°C. The supernatant was retained and centrifuged at 15,000 rpm for 1 h at 10°C. The pellet was resuspended in a minimal amount of buffer B (50 mM Tris–Cl, pH 7.9, containing 8 M urea, 1 mM EDTA, 1 mM DTT, 1 M NaCl, 0.5% NP-40, and 10% glycerol) and solubilized with a manual homogenizer. The homogenate was centrifuged in an SA600 rotor at 15,000 rpm for 1 h at 15°C. The supernatant was placed into 12,000 –14,000 MWCO dialysis tubing and dialyzed overnight against a 1:100 volume of buffer B without urea. The protein solution was then clarified by centrifugation in an SA600 rotor at 4000 rpm for 10 min at 4°C. |
| Enzyme activity,Kinetic analysis |
| None |
| Glycerol |
| 10% |
| n/a |
| n/a |
| Refolding temperature is not stated |