| Ampapathi RS, Creath AL, Lou DI, Craft JW Jr, Blanke SR, Legge GB.
(2008)
Journal of Molecular Biology,
1,
1 |
| Undefined |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 4 h |
| n/a |
| CTA1 protein expression and purification
CTA1 and CTA1-T2 were overexpressed in E. coli BL21(DE3) transformed with plasmids harboring the genes encoding these proteins. For unlabeled samples, E. coli BL21(DE3) was cultured in LB supplemented with 50 μg/mL of ampicillin. 15N-labeled and 15N/13C-labeled protein samples were generated by culturing E. coli BL21(DE3) in M9 minimal media supplemented with 50 μg/mL of ampicillin and containing 15NH4Cl as well as 15(NH4)2SO4 and 13C-labeled glucose (Sigma-Aldrich) as the sole nitrogen and carbon sources, respectively.
2H/15N/13C-labeled CTA1-T2 was generated by first culturing transformed E. coli BL21(DE3) in 10 mL of M9 minimal media supplemented with 50 μg/mL of ampicillin and containing 15NH4Cl, 15(NH4)2SO4, and 13C-labeled glucose. After incubation for 4 h at 37 °C, the cells were harvested by centrifugation at 4000 rpm in an F21B-8x50Y rotor (FIBERLite, Piramoon Technologies, Inc.); the available pellet was resuspended in M9 in 2H2O and added to 1 L of M9 in 2H2O with 50 μg/mL of ampicillin. Cells were cultured to mid log phase (with OD600 being approximately 0.6), expression was induced with 0.1 mM IPTG, and expression continued overnight at 37 °C. |
| IPTG |
| OD n/a =
n/a |
| Sonication |
| Lysozyme |
| None |
| insoluble |
| Column refolding: Nickel-chelating chromatography |
| 50 mM Tris, pH 8.0, 2 mM EDTA, 2 mM DTT, 200 μg/mL of lysozyme (Sigma-Aldrich), and 10% B-PER |
| 6 M Gdm–HCl, 20 mM Tris, pH 7.5, 200 mM NaCl, and 1 mM EDTA |
| 20 mM Tris, pH 7.5, 200 mM NaCl, and 1 mM EDTA |
| None |
| no tag |
| 7.5 |
| 1.0 |
| n/a |
| n/a |
| None |
| n/a |
| CTA1A2 was expressed as described for CTA1, and the harvested bacterial pellet was suspended in homogenization buffer. The cells were lysed by sonication; additional washes resulted in a clean inclusion body pellet. The pellet was solubilized in 6 M Gdm–HCl, 20 mM Tris, pH 7.5, 200 mM NaCl, and 1 mM EDTA. Renaturation and purification of the solubilized proteins were achieved by on-column refolding using the HisTrap™ HP affinity column (GE Healthcare) according to the manufacturer\'s instructions. The native refolding buffer was 20 mM Tris, pH 7.5, 200 mM NaCl, and 1 mM EDTA; elution from the column was achieved using 300 mM imidazole in 20 mM Tris, pH 7.5, and 200 mM NaCl. Further purification of the protein was completed using gel filtration chromatography essentially as described for CTA1. |
| Gel filtration chromatography |
| None |
| None |
| n/a |
| n/a |
| n/a |
| The refolding temperature is not specified |