| Legardinier S, Hubert JF, Bihan OL, Tascon C, Rocher C, Raguénès-Nicol C, Bondon A, Hardy S, Rumeur EL.
(2008)
Biochemica et Biophysica Acta,
1,
1 |
| Undefined |
| N-terminal +C terminal Glycine-serine dipeptide |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| ER2566 |
| 37.0 |
| 4 h |
| pGEX-4T1 |
| Boundaries of R1–3 and R20–24 proteins were chosen by the original alignment described by Winder [5]. In a first attempt, we tried to produce these two sub-domains including the irrespective hinges at the N- and C-terminal ends but the proteins precipitated. Finally, we decided to produce the proteins without any extension from the hinges. The construct corresponding to the single repeat R2 was from the group of W. Gratzer [8] and now known to have extensions of 8 residues at the N-terminal end and of 6 residues at the C-terminal end. By contrast, to improve its solubility, R23 was extended with 6 additional residues at the N-terminal end including the N-terminal glycine–serine dipeptide and 7 additional residues at the C-terminal end according to Kahana et al. [19] and [20] which recommended to extend the constructs by 6 to 8 residues. The Glutathione S-transferase (GST) gene fusion system was used to produce recombinant dystrophin rod domain R23 single repeat and R1–3 and R20–24 sub-domains, whose boundaries are indicated in Table 1. The pGEX-4T1 vectors with cDNAs encoding R23, R1–3 and R20–24 were transformed into the protease-deficient Escherichia coli ER2566 strain, and cultures were then grown to an absorbance at 600 nm of 0.5–0.8 at 37 °C in LB broth supplemented with 50 μg/mL ampicillin. Expression was induced by addition of 0.5 mM isopropyl-β-d-thiogalactopyranoside. After 4 h, bacteria were harvested by centrifugation at 4000 g for 15 min at 4 °C. Pellets were resuspended in 20 mL ice-cold lysis buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl with protease inhibitors) and incubated with 0.5 mg mL− 1 lysozyme on ice for 30 min before being broken up by sonication (U200S, UKA Labortechnic) on ice using 10 × 30 s cycles. |
| IPTG |
| OD 0.5-0.8 =
600 |
| Sonication |
| Lysozyme |
| Ion-exchange chromatography |
| insoluble |
| Dilution |
| 20 mM Tris–HCl, pH 7.5, 150 mM NaCl with protease inhibitors |
| EDTA 0.1 mM and NaCl 150 mM buffered with TRIS–HCl 20 mM at pH 7.5 |
| 8 M urea,EDTA 0.1 mM and NaCl 150 mM buffered with TRIS–HCl 20 mM at pH 7.5 |
| Ion-exchange chromatography |
| yes |
| 7.5 |
| 25.0 |
| 0.3–3 μM |
| n/a |
| None |
| n/a |
| Refolding kinetics
Stopped-flow data were recorded on a BioLogic (Grenoble, France) SFM-3, MOS-250 instrument. The dead time of the stopped-flow device was 2.2 ms. For refolding measurements, stock solutions of 8 M urea-treated proteins were prepared in TNE buffer. The refolding reaction was initiated at 25 °C by ten-fold dilution of the urea-treated protein with TNE buffer. The final protein concentrations were 0.3–3 μM. The time-dependent fluorescence of tryptophan changes were monitored at excitation and emission wavelengths of 295 nm and 350 nm, respectively. Curves used to determine rate constants were the averages of at least ten individual kinetic data points. Results were analysed by Biokine software (BioLogic). |
| Unspecified |
| None |
| None |
| n/a |
| n/a |
| n/a |
| n/a |