| Pungercar J, Krizaj I, Liang NS, Gubensek F.
(1999)
Biochem J.,
341,
139-145 |
| Undefined |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 3 h |
| pALTER-1 |
| Expression in E. coli
The E. coli strain BL21(DE3) (Novagen) was transformed with the expression plasmid containing the Atx gene. For large-scale production, a single fresh bacterial colony was used to inoculate 5 ml of Luria–Bertani medium supplemented with 100 ug/ml ampicillin and the cells were grown until the culture became turbid. The culture was added to 500 ml of rich medium
containing 10 g\\l tryptone, 5 g\\l yeast extract, 5 g/l NaCl, 7.5 g/l Na
2HPO4, 3 g/l KH2PO4, 1 g/l NH4Cl, 4 g/l glucose and 1 mM MgSO4 (pH 7.0), in the presence of 100 mg/l ampicillin. The preculture was grown overnight and used to inoculate 7 l of the same medium in a 10-l fermentor (New Brunswick). Bacteria were grown under vigorous aeration and agitation at 37 C. Production of recombinant toxin was induced in mid-log phase
(D600= 2.4) by adding isopropyl β-D-thiogalactoside to a final concentration of 0.3 mM. Approx. 3 h after induction, when the culture became saturated (D600= 5–6), the cells were harvested by centrifugation at 4 C. Typically, about 10 g of wet bacterial cells per l of fermentation broth were obtained. |
| IPTG |
| OD n/a =
n/a |
| Sonication |
| Lysozyme |
| not specified |
| insoluble |
| Dialysis |
| 100 ml of buffer containing 0.5 M urea, 50 mM Tris/HCl and 5 mM EDTA (pH 8) |
| 7 M guanidine hydrochloride\\0.35 M Na2SO4(pH 8.3) |
| 25 mM boric acid (pH 8.0) containing 10 mM CaCl2, 8 mM cysteine, 1 mM cystine and 1 mM EDTA, in the presence of 0.5–2 M denaturant ; either guanidine hydrochloride (0.5–1 M) or urea (1–2 M) |
| not specified |
| no tag |
| 8.0 |
| 4.0 |
| n/a |
| 16-20 h |
| Cysteine/Cystine |
| 8/1 mM |
| Isolation of inclusion bodies and refolding of proteins The bacterial pellet from a 7-l culture was resuspended in 300 ml of ice-cold TE buffer [50 mM Tris/HCl/40 mM EDTA (pH 8.0)] containing 25 % (w\\v) sucrose and homogenized. After adding of 50 mg of lysozyme, the suspension was incubated for 60 min on ice with occasional homogenization. TE buffer (300 ml) was then added and the suspension allowed to stand for 30 min on ice
with periodic homogenization. The cells were sonicated three times on ice in 100-ml portions for 1.5 min each. In cases where the lysate still remained too viscous, 1 mg of DNase I was added and the sample was incubated for about 30 min at room temperature. After reduction of viscosity, 0.1 % (w\\v) Triton X- 100 was added and the lysed cells were homogenized for a few seconds and sonicated three times for 1 min each. The lysate was centrifuged for 30 min at 4000 g, the pellet thoroughly washed with 100 ml of buffer containing 0.5 M urea, 50 mM Tris/HCl and 5 mM EDTA (pH 8), and the inclusion bodies recovered by centrifugation using the same conditions. The inclusion bodies were dissolved in 150 ml of a freshly prepared solution of 7 M guanidine hydrochloride/0.35 M Na2SO4 (pH 8.3). The fusion protein was S-sulphonated as described by Thannhauser and Scheraga [20] and precipitated subsequently by adding ice-cold glacial acetic acid to a final concentration of 1 % (v/v). The sulphonated protein was pelleted by centrifugation for 30 min at 5000 g, dissolved in 8 M urea or 5 M guanidine hydrochloride (pH 8.0), and stored at -20 C until use. Refolding was performed for 16–20 h at 4 mC in 25 mM boric acid (pH 8.0) containing 10 mM CaCl2, 8 mM cysteine, 1 mM cystine and 1 mM EDTA, in the presence of 0.5–2 M denaturant ; either guanidine hydrochloride (0.5–1 M) or urea (1–2 M). Typically, the protein concentration was about 75 µg/ml in 2 l of renaturation solution.
|
| Enzyme activity,SDS-PAGE,Circular Dichroism (uv-CD) |
| None |
| None |
| n/a |
| 25 % |
| 2-5 mg/ |
| n/a |