| Ito M, Nagata K, Kato Y, Oda Y, Yamagoe S, Suzuki K, Tanokura M.
(2003)
Protein Expression and Purification,
27,
272-278 |
| Over expression & Renaturation |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 6 h |
| pET21a |
| Escherichia coli strain BL21(DE3) (Novagen) harboring the expression vector of (His)6-LECT2 [9] based on the plasmid pET-21a(+) (Novagen) was cultivated in 1.5 L LB medium using a 5-L Erlenmeyer flask at 37 °C to an OD600 of 0.8. The cells were then collected by centrifugation at 5000g for 5 min at room temperature, resuspended in 1.5 L of a modified M9 medium, which contained 20 g KH2PO4, 15 g K2HPO4, 34 g Na2HPO4·12H2O, 3.6 g K2SO4, 1.0 g NH4Cl, 1.0 g glucose, 290 mg MgCl2, 45 mg thiamine, and 15 ml of 100× Basal Medium Eagle vitamin solution (Gibco-BRL Life Technologies), and cultivated again at 37 °C. At an OD600 of 0.8, 360 mg IPTG (to a final concentration of 1.0 mM), 1.0 g NH4Cl, and 1.0 g glucose were added and the culture was continued at 37 °C for 6 h. The cells were then harvested by centrifugation at 10,000g for 3 min at 4 °C. |
| IPTG |
| OD 0.8 =
600 |
| Not stated |
| None |
| Ni-NTA agrose chromatography |
| insoluble |
| Column refolding: Nickel-chelating chromatography |
| n/a |
| 0 mM Tris–HCl (pH 7.9), 5 mM imidazole, and 500 mM NaCl] that contained 8 M urea and 10 mM DTT |
| 20 mM Tris–HCl (pH 7.9), 20 mM imidazole, 500 mM NaCl, and 20% glycerol,reduced and oxidized glutathione was added to a final concentration of 1 mM each |
| Ni-NTA agrose chromatography |
| no |
| 7.9 |
| 4.0 |
| n/a |
| 7 days |
| GSH/GSSG |
| 1/1 mM |
| Affinity purification and refolding of (His)6-LECT2
All of the procedures in this section were carried out at 4 °C.
Step 1. The collected cells were resuspended in 50 ml buffer A [20 mM Tris–HCl (pH 7.9), 5 mM imidazole, and 500 mM NaCl] that contained 8 M urea and 10 mM DTT. This suspension was then shaken gently overnight. After centrifugation at 45,000g for 30 min, 450 ml of 6 M urea in buffer A was added to the supernatant to lower the concentration of DTT to 1 mM or less. To this diluted supernatant, 10 ml of a 50% slurry of Ni-NTA agarose (Qiagen) was added and suspended by gentle stirring for 1 h. The suspension was then transferred to an empty column (15-mm inner diameter, Bio-Rad) and the flow-through was discarded. The column-packed Ni-NTA resin with bound proteins was washed first with 20 ml of 6 M urea in buffer A and then with 25 ml of 2 M urea in buffer B [20 mM Tris–HCl (pH 7.9), 20 mM imidazole, 500 mM NaCl, and 20% glycerol]. The urea concentration was then lowered from 2 to 1 M using a linear gradient over 20 h at a flow rate of 0.1 ml/min.
Step 2. The resin was transferred to a 1-L polypropylene beaker (Nalgene) and suspended in 150 ml of 1 M urea dissolved in buffer B with gentle stirring. To this suspension, the reduced and oxidized glutathione was added to a final concentration of 1 mM each and the suspension was stirred gently for 7 days. The resultant suspension was transferred evenly into two empty columns (15 mm i.d., Bio-Rad) and the flow-through was discarded. The proteins bound to the resin were eluted with 10 ml in buffer C [20 mM Tris–HCl (pH 7.9), 1 M imidazole, 500 mM NaCl, and 20% glycerol] containing 1 M urea for each column.
Step 3. The protein concentration in the eluate was determined with a Protein Assay Kit (Bio-Rad) that used the biuret reaction. The eluate was transferred to a 1-L polypropylene beaker and diluted with 1 M urea in buffer B to lower the protein concentration to 10 μg/ml, and was stirred gently for 7–10 days to complete the disulfide bond formation. |
| SDS-PAGE |
| None |
| Glycerol |
| 20% |
| 1.0 mg/L |
| n/a |
| n/a |