| Feldmar S, Kunze R.
(1991)
EMBO J,
10,
4003-4010 |
| Undefined |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| HMS174 |
| 32.0 |
| overnight |
| pET-3 |
| Preparation of inclusion bodies
Fifty ml ZB and 200 ytg/ml ampicillin were inoculated with a single colony
and grown at 32°C overnight. Prewarmed M9ZB (1 litre), 200 /Ag/ml
ampicillin were inoculated with 2.5 ml overnight culture and shaken at 37°C. After 2 h 2 ml ampicillin (100 mg/mi) were added. At an OD6W -0.6
the culture was induced with IPTG (0.4 mM) and another 2 ml ampicillin
(100 mg/mi) were added. The cells were harvested 2-3 h after induction
by centrifugation. One to 3 g cells were resuspended in 15 ml buffer I
(10 mM Tris-Cl, 1 mM EDTA, 0.2% Triton X-100, pH 8.3), adjusted
to 6 mM MgCl2 and 15 U/ml Benzonase (Merck), and homogenized twice
in a French Press under high pressure. Protease inhibitors (0.5 mM PMSF,
1 Agg/ml each of aprotinin, leupeptin, pepstatin A and antipain) were added
to the lysate, followed by a 15-30 min incubation at 4°C, and 25 min
centrifugation at 20 000 g (4°C). The pellet was resuspended in 20 ml buffer 1 (20 mM Tris-Cl, 0.5 M NaCl, 5 mM EDTA, 0.5% Triton X-100, 10%
glycerol) with protease inhibitors and centrifuged for another 25 min at
15 000 g (4°C). The resulting sediment contains the inclusion bodies. They
were suspended in 5 ml buffer II and, after protein concentration
determination with the Micro BCA reagent (Pierce), adjusted to 5 mg/ml.
|
| IPTG |
| OD 0.6 =
600 |
| French Press |
| Detergents |
| Size-exclusion chromatography |
| insoluble |
| Dilution |
| 20 mM Tris-Cl, 0.5 M NaCl, 5 mM EDTA, 0.5% Triton X-100, 10% glycerol |
| 6 M guanidinium chloride, 0.1 M DTT, 100 mM Tris-Cl, 2 mM EDTA, pH 8.5 |
| 50 mM Tris-Cl, 50 mM NaCl, 3 mM MgCl2, 5 AM ZnC12, 0.2% Triton X-100, 5 mM glutathione-reduced, 0.5 mM glutathione-oxidized, 10% glycerol, pH 8.5 |
| Size-exclusion chromatography |
| no tag |
| 8.5 |
| 4.0 |
| n/a |
| overnight |
| GSH/GSSG |
| 5/0.5 mM |
| Chromatographic purification
Recombinant protein was purified from a contaminating nuclease activity
by size-exclusion chromatography in a denaturing solvent. Inclusion bodies
were dissolved by shaking for at least 1 h at room temperature in denatura-
tion buffer (6 M guanidinium chloride, 0.1 M DTT, 100 mM Tris-Cl,
2 mM EDTA, pH 8.5). After removal of insoluble material by centrifuga-
tion, - 1 mg protein was applied to an FPLC-Superose-12 column
(Pharmacia) and size fractionated at 0.4 ml/min in denaturation buffer. Eluted fractions (0.25 ml) were analysed by SDS-PAGE. Individual fractions
containing ORFa protein (derivatives) were renatured as described below.
Renaturation Renaturation of the bacterial proteins was performed essentially as described by Jaenicke and Rudolph (1989). Aliquots of the inclusion body suspension were sedimented by centrifugation and dissolved by shaking for at least 1 h at room temperature in denaturation buffer to a final concentration of 2.5 mg/ml. After removal of insoluble components by centrifugation, aliquots were diluted 1:100 or 1:50 with ice-cold renaturation buffer (50 mM Tris-Cl, 50 mM NaCl, 3 mM MgCl2, 5 AM ZnC12, 0.2% Triton X-100, 5 mM glutathione-reduced, 0.5 mM glutathione-oxidized, 10% glycerol, pH 8.5) and incubated overnight at 4°C.
|
| Mobility shift assays |
| None |
| Triton X-100 |
| 0.2% |
| n/a |
| n/a |
| n/a |