| Calero M, Mendez E, Garcia E
(1995)
Biochim Biophys Acta.,
1249,
91-99 |
| Recombinant Protein Expression |
| C-LytA |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| TG1 |
| 37.0 |
| overnight |
| pCUZ2HC |
| E coli TG1 harbouring plasmid pCUZ2HC was grown ON at 37C in LB medium containing ampicillin (10ug/ml). It should be pointed out that the addition of lac inducer such as ITPG was unnecessary since no repression of the lpp-5-lac promoter could be detected, probably due to the high copy number of pCUZ2HC. Cells were harvested by centrifugation, washed with 20 mM sodium phosphate buffer (pH 6.9), and lysed by two passages through a French pressure cell. The fusion protein in the pellet was recovered by centrifugation (7500 x g, 10 min, 4 C) and the pellet washed three times at room temperature with 3 M urea in 0.1 M Tris-HCl (pH 8) with magnetic stirring. |
| Not Stated |
| OD n/a =
n/a |
| French Press |
| None |
| None |
| insoluble |
| Dilution/Dialysis combination |
| 3 M urea, 0.1 M Tris-HCl (pH 8) |
| 6 m guanidinium chloride, o.1 M Tris-Hcl buffer, 2 mM EDTA, 0.3 M dithioerythrithol, (ph 8.0) |
| 0.1 M Tris-HCl (pH 8), 0.5 M L-arginine, 8 mM oxidized glutathion, 2 mM EDTA |
| None |
| no |
| 8.0 |
| 10.0 |
| n/a |
| 24-48 h |
| DTT |
| 0.3 M,0.3 M |
| The washed inclusion bodies were resuspended in denaturation buffer (6 M guanadinium chloride, 0.1 M Tris-HCl buffer, containing 2 mM EDTA and 0.3 M DTT, pH 8) and incubated with shaking for 2 h at room temp. Afterwards the insoluble pellet was removed by centrifugation (30000 x g, 30 min, 4 C) the supernatant was dialyzed exhaustively against 0.1 M Tris-HCl (pH 8) and cetrifuged again. |
| Immunoassay |
| None |
| None |
| n/a |
| n/a |
| n/a |
| Protein was a C-LytA/HC fusion |