| Ward RJ, de Oliveira AH, Bortoleto RK, Rosa JC, Faca VM, Greene LJ
(2001)
Protein Expression and Purification,
21,
134-140 |
| Undefined |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21 |
| 37.0 |
| 5h |
| pET11-d |
| One-hundred fifty milliliters of growth medium (2.5g yeast extract; 10 mM MgSO4, pH 7.5; 15 mg ml21 chloramphenicol; 150 mg ml21 ampicillin) was inoculated with E. coli strain BL21(DE3){pLysS} transformed with pET11d 1 BTXI and grown at 37degC to an A600 of 0.6. Recombinant protein expression was induced by addition of 0.6 mM IPTG, and the culture was grown for an additional 5 h.
|
| IPTG |
| OD 600 =
0.6 |
| Sonication |
| None |
| Size-exclusion chromatography |
| insoluble |
| Column refolding: Size-exclusion chromatography |
| 50mM Tris–HCl pH 8.0, 1 mM EDTA, 1% Triton X-100 |
| 4 M GdnSCN, 2 mM EDTA, 300 mM Na2SO3, 50mM NTSB |
| 50 mM TrisHCl, pH 8.0; 1 mM EDTA, 2 mM oxidized glutathione; 4 mM reduced glutathione; 4 mM n-dodecyl-b-D-maltoside |
| Size-exclusion chromatography |
| no tag |
| 8.0 |
| 25.0 |
|
| 16h |
| GSH/GSSG |
| 4mM/2mM |
| Inclusion bodies were solubilized in 0.6 ml of denaturation buffer containing sodium sulfite and the cysteine sulfonating compound 2-nitro-5-thiobenzoic acid (NTSB) (4 M GdnSCN, 2 mM EDTA, 300 mM Na2SO3, 50mM NTSB). The solubilized inclusion bodies were incubated for 30 min at 45degC to ensure complete reduction
and sulfonation of cysteine residues. The mixture was subsequently passed through a BioGel P6 column (2 x 3cm, Bio-Rad) previously equilibrated in buffer A (2 M guanidinium hydrochloride; 1 mM EDTA; 50 mM Tris?HCl, pH 8.0) to separate the modified protein from excess NTSB and to exchange the buffer. The protein peak, eluted in a total volume of approx. 0.8ml, served as the starting material for the refolding process. Columns (138 cm) packed with the BioGel P6 resin were equilibrated with 3 column vol of buffer B (50 mM Tris-HCl, pH 8.0; 1 mM EDTA), followed by 1 column vol of buffer C (buffer B containing 2 mM oxidized glutathione; 4 mM reduced glutathione; 4 mM n-dodecyl-b-D-maltoside). The protein was eluted at a concentration of 0.9mg/ml, and 0.8 ml of this solution was applied by gravity flow at 150-200 microliters/h. Typically sample application was complete in 4 h, after
which an additional 0.7 ml of buffer C was applied at the same rate. After incubation for 12 h at 25degC and the protein was eluted with 2 column vol of buffer B. The eluate (12 ml) was collected in a 15-ml stoppered polypropylene tubes, sealed, and incubated for an additional 36 h at 25degC with mild agitation. Since a slight flocculent precipitate was observed, the incubation product was passed through a 0.45-mm cellulose acetate/cellulose nitrate filter. |
| Enzyme activity |
| None |
| None |
|
| 5.7% - 4-5mg/L culture |
|
|