| Lee SH, Carpenter JF, Chang BS, Randolph TW, Kim YS.
(2006)
Protein Science,
15,
304-13 |
| Protein refolding |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 6 h |
| pProExHTc |
| Protein expression and purification of IBs
The bacterial expression plasmids encoding the extracellular domain (ECD) of GNBPs were provided by Dr. B.H. Oh (Pohang University of Science and Technology). Briefly, residues 24–492 of GNBP1-ECD and residues 25–461 of GNBP2-ECD were cloned on pProEx-HTb (Invitrogen) using BamHI/XhoI sites, and residues 31–483 of GNBP3-ECD was subcloned into pProExHTc (Invitrogen) using EcoRI/XhoI sites. For the bacterial expression of the intracellular phosphatase domain of PTPRS and DUSP7, residues 1386–1948 of PTPRS and residues 1–130 of DUSP7 were subcloned onto pET28a (Novagen) using NdeI/BamHI sites, which were provided by Dr. J.S. Kim (Korea Research Institute of Bioscience and Biotechnology). E. coli strain BL21(DE3) (Novagen) was used as an expression host. Cells were grown at 37°C to an OD600 of ~0.8 in 100 mL of Luria-Bertani medium containing 100 µg/mL amplicillin, and protein expression was induced by the addition of 1 mM isopropyl--D-1-thiogalactoside. After a 6-h induction at 37°C, cells were harvested by centrifugation at 12,000g for 10 min at 4°C and resuspended in a 10-mL lysis buffer (50 mM Tris-Cl at pH 8.0, 5 mM EDTA, 100 mM NaCl, 1 mM PMSF). IBs were purified by following the procedure described previously (Bowden et al. 1991; De Bernardez Clark et al. 1999). The purified IBs were washed three times with double distilled water, and stored at –80°C until used. The contents of the expressed protein in purified IBs were more than 70% based on SDS-PAGE analyses (Fig. 1). After gel staining with Coomassie blue, the intensity of bands corresponding to monomeric proteins in the reducing and nonreducing SDS-PAGE was compared with estimate fractions of monomeric species in the nonreducing condition using an image analysis system (Model GS-700, Bio-Rad). |
| IPTG |
| OD 0.8 =
600 |
| Not stated |
| None |
| not specified |
| insoluble |
| High Pressure |
| double distilled water |
| 50 mM Tris-Cl at pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.05% sodium azide |
| 50 mM Tris-Cl at pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.05% sodium azide2 mM DTT and 6 mM GSSG |
| not specified |
| no tag |
| 8.0 |
| 25.0 |
| n/a |
| 24 h |
| GSSG/DTT |
| 6/2 mM,6/2 mM,6/2 mM,6/2 mM |
| Bs were resuspended in a TBSE buffer (50 mM Tris-Cl at pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.05% sodium azide), containing a redox-shuffling mixture (2 mM DTT and 6 mM GSSG), designated as a \"refolding buffer\". The molar ratio of oxidized to reduced glutathione (GSSG:GSG) was 1:1 because the mixture of 2 mM DTT and 6 mM GSSG rapidly converts to 4 mM GSH and 4 mM GSSG, with a byproduct of 2 mM oxidized DTT (St. John et al. 2002). For testing the effects of additives, urea (1 and 2 M), L-arginine (0.5 M), glycerol (2.5 M), Tween 20 (0.1 mM), or Triton X-100 (0.5 mM) were added to the refolding buffer. The critical micelle concentrations (CMCs) of Tween 20 (Sigma T8787) and Triton X-100 (Sigma P5927) provided by the supplier (Sigma) were 0.06 and 0.32 mM in water, respectively.
Samples (~300 µL) were placed in sterile, disposable 1-mL syringes (one end heat sealed and the other sealed with the rubber plunger) and placed in a custom-made high-pressure vessel (St. John et al. 2001; Kim et al. 2005). The vessel was sealed and pressurized with a high-pressure crank generator (rated up to 700 MPa) from High Pressure Equipment Co., using water as a pressure transmitting fluid (St. John et al. 2001; Kim et al. 2005). Samples were slowly pressurized (10 min) to the desired pressure and held there for 24 h. Unless otherwise specified, samples were depressurized at 10 MPa per 10 min. All pressure experiments were performed at room temperature (25°C). |
| Activity assay |
| None |
| L-Arginine,Glycerol,Triton X-100,tween 20 |
| 2.5/0.5/0.5/0.1 M |
| n/a |
| n/a |
| n/a |