| Kwasek A, Osmark P, Allhorn M, Lindqvist A, Kerstrom B, Wasylewski Z.
(2007)
Protein Expression and Purification,
53,
145-52 |
| Undefined |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3)pLysS |
| 25.0 |
| 4-5 hrs |
| pET 12a |
| Freshly transformed E. coli BL21(DE3)pLysS were grown in LB medium supplemented with ampicillin (Amp, 100 ug/ml) and chloramphenicol (CAM, 34 ug/ml). Protein expression was induced with 1 mM IPTG when OD600 reached 1.1–1.3 and the bacteria were cultured for additional 4–5 h. Induction with IPTG at higher OD600 levels (i.e. near and above 1.1 value rather than below 0.9) was observed to give higher amounts of the protein and better results during purification procedures. The cells were harvested (20 min at 8000g, 4 degC), resuspended in 0.5 l of the column buffer and sonicated on ice for 1 min at maximum level using Sonifier B-12 cell disruptor (Branson Sonic Power Company). Following centrifugation (15 min at 8000g), cells were resuspended in 50 ml of the column buffer, sonicated as previously and incubated at room temperature for 0.5 h with DNase and RNase (10 microg/ml each). The pellet (harvested for 20 min, 15,000g) was dissolved in 50 ml of GuHCl buffer and left at room temperature for at least 1 h. |
| IPTG |
| OD 600 =
1.1 |
| Sonication |
| None |
| Metal affinity chromatography |
| insoluble |
| Dilution |
| 0.5 M NaCl, 20 mM Tris–HCl, pH 8.0 |
| 6 M guanidinium chloride, 20 mM Tris–HCl, pH 8.0 |
| 0.5 M arginine, 0.65 M NaCl, 2 mM EDTA, 0.1 M Tris–HCl, pH 8.0 |
| Metal affinity chromatography |
| no |
| 8.0 |
| 4.0 |
| n/a |
| Overnight |
| GSH |
| 5 mM,5 mM |
| The protein extract was then centrifuged twice (30 min, 20,000g) and applied on a Ni-NTA column equilibrated with GuHCl buffer. The resin was washed with 5 bed volumes of GuHCl buffer and the bound proteins were eluted with 0.5 M imidazole. Fractions containing ?1m were mixed with a water solution of reduced glutathione (half-volume of the pooled fractions), dripped into 100–150 ml of the folding buffer containing oxidized glutathione, while stirring and left overnight at 4 degC, without stirring. Final concentrations of the reduced and oxidized glutathione were of 5 and 1 mM, respectively. The sample was then dialysed twice against 2 l of the column buffer, repurified on a Ni-NTA column equilibrated with the column buffer and dialysed thoroughly against Tris buffer. Human plasma and urine ?1m were purified as described previously [9] and [17]. To determine protein concentration A0.1% coefficients at 278 nm of 1.49, 1.6 and 1.8 were used for recombinant, plasma and urine proteins, respectively. |
| Ultraviolet (UV) Absorbance,Far-UV Circular Dichroism,Fluorescence,Immunoassay,SDS-PAGE,Gel filtration chromatography |
| None |
| L-Arginine |
| 0.5 M |
| 40-50 mg/litre |
| >95% |
| n/a |