| Küster F, Seckler R.
(2008)
Biochemistry,
1,
1 |
| Folding |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 30.0 |
| overnight |
| pET21a |
| Preparation of PslS. The fragment coding for Thr 1 to Gln 241 of Psl (with the termini corresponding to the isoform B of PslP (12)) was amplified from pMP2809 (13), ligated into the expression plasmid pET21a (14), and Escherichia coli BL21 (DE3) cells were transformed with the resulting vector.
For purification, a protocol adapted from (15), (16) and (13) was used. Bacteria were grown in LB full medium (5 L), induced for protein production at an optical density of 1 at 550 nm, and cells were harvested by centrifugation after overnight incubation at 30 C. |
| Not Stated |
| OD 1 =
550 |
| High pressure homogenization |
| None |
| None |
| insoluble |
| Dilution |
| 10 mM Tris/HCl, 150 mM NaCl, 0.5 M Pefabloc SC, pH 6.8 |
| 8 M GdmCl, 10 mM Tris, pH 6.8 |
| 10 mM Tris/HCl, 150 mM NaCl, 1 mM CaCl2, 1 mM MnCl2, pH 6.8, 1.5 M urea |
| None |
| no tag |
| 6.8 |
| 4.0 |
| n/a |
| overnight |
| Not specified |
| n/a |
| The frozen pellets were resuspended in 20 mL of 10 mM Tris/HCl, 150 mM NaCl, 0.5 M Pefabloc SC, pH 6.8, and cells were disrupted by high-pressure lysis. The insoluble fraction was suspended in 30 mL of solubilization buffer (8 M GdmCl, 10 mM Tris, pH 6.8) and gently shaken overnight at 4 C. The supernatant was diluted 1:200 with ice-cold refolding buffer (10 mM Tris/HCl, 150 mM NaCl, 1 mM CaCl2, 1 mM MnCl2, pH 6.8) containing 1.5 M urea and stirred overnight at 4 C. The renatured protein was concentrated by ammonium sulfate precipitation and further purified by affinity chromatography as described (11). Purity of PslP and PslS was tested by SDS gel electrophoresis with silver staining. The purified proteins were stored at -70 C. |
| SDS-PAGE |
| None |
| None |
| n/a |
| n/a |
| n/a |
| n/a |