| Chen XG,Gong Y,Li H,Lun ZR,Fung MC
(2001)
Protein Expression and Purification,
23,
33-37 |
| Recombinant Protein Expression |
| C-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 3 h |
| pET30a(+) |
| Expression in E. coli
A single colony of E. coli BL21(DE3), harboring the plasmid pET-30a(􏰉)-􏰅SP30􏰅PI, was grown in LB medium (10 g tryptone, 5 g yeast extract, and 10 g NaCl per liter of medium) plus 100 􏰈g/ml ampicillin overnight at 37􏰋C and then diluted 10-fold with fresh LB medium. Production of SAG1 was induced with IPTG at a final concentration of 1.0 mM when the absorbance of culture at 600 nm reached 0.5. The cells were harvested by
centrifugation after 3 h of induction.
|
| IPTG |
| OD 0.5 =
600 |
| Freeze-thaw |
| None |
| Metal affinity chromatography |
| insoluble |
| Dilution/Dialysis combination |
| 1 M urea, 0.5% Triton X-100, 50 mM Tris –HCl, pH 8.0, 1 mM EDTA |
| 50 mM NaH2PO4, pH 8.0, 10 mM Tris –HCl, pH 8.0, 8 M urea, with 0.05% Tween |
| dilution buffer (50 mM NaH2PO4, pH 8.0, 100 mM NaCl), 0.5 M urea, 20 mM Tris –HCl, pH 8.0, 1 mM EDTA |
| Metal affinity chromatography |
| no |
| 8.0 |
| 4.0 |
| n/a |
| 24 h |
| None |
| n/a |
| Refolding. The eluted 6􏰊 His –SAG1 or solubilized inclusion bodies were diluted with dilution buffer (50 mM NaH2PO4, pH 8.0, 100 mM NaCl) to obtain a final concentration of protein of 50–100 􏰈g/ml. The concentration of urea was approximately 0.5–2 M. The sample was dialyzed first against buffer I (0.5 M urea, 20 mM Tris –HCl, pH 8.0, 1 mM EDTA) at 4C for 24 h and
then against buffer II (20 mM Tris –HCl, pH 8.3, 1 mM EDTA, 2 mM reduced glutathione, 0.2 mM oxidized glutathione) at 4C for 24 h.
|
| ELISA |
| None |
| None |
| n/a |
| n/a |
| n/a |
| n/a |