| Levy I, Shoseyov O
(2001)
J Pept Sci.,
7,
50-57 |
| Protein refolding |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 4 h |
| pET-29a |
| Expression of HRP/tHRP in E. coli
HRP and tHRP were overexpressed in E. coli BL21 (DE3) harbouring the pET29a-HRP or pET29a-tHRP plasmids. Inoculum was prepared by growing a single colony in 4 ml LB (10 g/l bactotrypton, 5 g/l yeast extract, 10 g/l NaCl) medium containing 50 mg/ml kanamycin at 37°C to an OD600 of 0.6 – 1.0. After adding 2 ml inoculum to 100 ml fresh LB medium containing 50 mg/ml kanamycin, cells were grown in shaking flasks at 250 rpm and 37°C to an OD600 of 0.6, after which 1 mM (final concentration) isopropyl-D-thiogalactopyranoside (IPTG) was added. Following 4 h of incubation, the cells were harvested by centrifugation at 1600 g for 10 min, and washed twice in 20 mM Tris pH 8.0. The bacterial pellet was resuspended in 30 ml lysis buffer (20 mM Tris pH 8.0, 1 mM EDTA, 0.1% (v/v) Triton, 10 mg/ml lysozyme, 5 mg/ml DNaseI, 0.5 mM phenyl methyl sulphonyl fluoride) and incubated at 37°C for 30 min. Inclusion bodies were collected by centrifugation at 15 000 g for 10 min followed by four washes in 20 ml 20 mM Tris pH 8.0 containing 1 mM EDTA and 1% Triton.
|
| IPTG |
| OD 0.6 =
600 |
| None |
| Lysozyme |
| None |
| insoluble |
| Dialysis |
| 20 ml 20 mM Tris pH 8.0 containing 1 mM EDTA and 1% Triton |
| 4.5 M urea, 40 mM Tris base pH 11.3 and 1 mM Cys |
| 20 mM Tris base pH 8.5 containing 0.5 M urea, 0.2 mM hemin, 5 mM CaCl2 and 150 mM oxidized glutathione (first dialysis), and then for 5 h at 4°C against 20 mM Tris base pH 8.5 in 50% (v/v) glycerol containing 5 mM CaCl(second dialysis) |
| None |
| no tag |
| 8.5 |
| 4.0 |
| 0.1 mg/ml |
| 10 h |
| GSSG |
| 150 uM |
| Refolding of HRP
Inclusion bodies were dissolved in urea solution (4.5 M urea, 40 mM Tris base pH 11.3 and 1 mM Cys) to a protein concentration of 0.1 mg/ml, as determined by bicinchoninic acid protein assay (Pierce, Rockford, IL, USA), and stirred for 1 h to solubilize the inclusion bodies. The denatured proteins were dialysed for 5 h at 4°C against 20 mM Tris base pH 8.5 containing 0.5 M urea, 0.2 mM hemin, 5 mM CaCl2 and 150 mM oxidized glutathione (first dialysis), and then for 5 h at 4°C against 20 mM Tris base pH 8.5 in 50% (v/v) glycerol containing 5 mM CaCl2 (second dialysis). The protein solution was analysed by 12.5% SDS-PAGE [21]. Specific activity was determined according to Josephy et al. [22] using 3,3,5,5-tetramethylbenzidine (TMB) as a substrate.
|
| Unspecified,SDS-PAGE,Protein activity assay |
| None |
| Glycerol |
| 50% v/v |
| n/a |
| n/a |
| n/a |