| Zhang Q, Horst R, Geralt M, Ma X, Hong WX, Finn MG, Stevens RC, Wüthrich K.
(2008)
J Am Chem Soc,
1,
1 |
| Structural Studies |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3)pLysS |
| 37.0 |
| 4 h |
| pET-3b |
| Expression and Purification of OmpX in D2O Medium. OmpX was precloned in the pET 3b plasmid and transformed into E. coli BL-21 (DE3) pLysS (Stratagene) competent cells for expression. One colony was used to inoculate a culture flask containing 20 mL of LB broth with the necessary antibiotics and shaken at 37 °C overnight. The cell culture was adapted to D2O by inoculating 4 mL of standard M9 minimal medium containing 33% v/v D2O with 40 µL of this LB culture and shaken at 37 °C overnight. 100 µL of the 33% D2O culture were then used to inoculate 10 mL of M9 minimal medium with 66% v/v D2O and similarly shaken overnight. A uniformly 2H,15N-labeled OmpX sample was prepared by inoculating 1 L of standard M9 minimal medium containing 99% D2O (Spectra Gases) and 1 g/L of [15N]-ammonium chloride (CIL) with the resulting 10 mL of D2O-adapted culture. This culture was shaken at 37 °C until the cell density reached an OD600 of approximately 1.0, and protein expression was then induced with 1 mM isopropyl-α-D-thiogalactopyranoside. Upon reaching the stationary growth phase after approximately 4 h, the cells were harvested at 5000 g for 10 min. TE buffer (20 mM Tris-HCl at pH = 8.0, 5 mM EDTA) was used to resuspend the cell pellet in a volume corresponding to approximately 10 times the wet cell mass in grams and sonicated for 25 min using a Misonix 3000 sonicator. The solution was pelleted at 4300 g for 1 h, and the supernatant was discarded. The remaining insoluble pellet was resuspended in the same volume of TE buffer plus 2% (v/v) Triton X-100 at room temperature, pelleted at 4300 g for 30 min, and the supernatant was discarded. Triton was then removed by repeating the same procedure with TE buffer. OmpX was recovered from the inclusion bodies by resuspending the cell debris in TE buffer plus 6 M urea for 2 h at room temperature. Remaining cellular debris were then pelleted at 48 000 g for 20 min at 4 °C, and the unfolded OmpX in the supernatant was recovered for ion exchange purification. |
| IPTG |
| OD 1.0 =
600 |
| Sonication |
| None |
| Ion-exchange chromatography |
| insoluble |
| Dilution into detergent |
| TE buffer (20 mM Tris-HCl at pH = 8.0, 5 mM EDTA) |
| TE buffer plus 6 M urea |
| 20 mM Tris-HCl at pH = 8.5, 5 mM EDTA, 600 mM L-Arg, and 2% (w/v) PMAL-C10 |
| Ion-exchange chromatography |
| no tag |
| 8.5 |
| 4.0 |
| n/a |
| 4 h |
| None |
| n/a |
| A previously established protocol16 for OmpX reconstitution in DHPC (Avanti Polar Lipids) detergent micelles was modified for microscale experiments with a variety of widely different detergents (Figure 1). At 4 °C, 100 µL of unfolded OmpX at a concentration of 10 mg/mL were added to 600 µL of refolding buffer (20 mM Tris-HCl at pH = 8.5, 5 mM EDTA, 600 mM L-Arg, and 2% (w/v) detergent) over a period of 4 h with vigorous stirring. The resulting OmpX solution was vigorously stirred at 4 °C for ~16 h. The detergent-refolded OmpX was then collected and exchanged into NMR buffer (20 mM sodium phosphate at pH = 6.8, 100 mM NaCl, 0.3% NaN3, 10% D2O, detergent) by way of concentration and dilution using Vivaspin 500 concentrators (10 000 molecular weight cutoff), alternatively using NMR buffer with and without 2% (w/v) of detergent. The final sample was concentrated to 50 µL. |
| NMR analysis |
| None |
| L-Arginine,Detergents |
| 2% v/W/ 600mM |
| <5% |
| n/a |
| The detergent is PMAL-C10 |