| Hrzenjak A, Frank S, Maderegger B, Sterk H, Kostner GM.
(2000)
Protein Engineering,
13,
661-666 |
| Over expression & Renaturation |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 4 h |
| pT7-7 |
| The recombinant protein was expressed in E.coli strain BL-21 (DE3) under the following conditions: 1 l of LB medium [10 g of tryptone (Difco), 5 g of yeast extract (Difco), 5 g of NaCl/l distilled H2O, pH 7.4] containing 50 µg/ml ampicillin (Sigma) was inoculated with 10 ml of overnight culture of BL-21 (DE3) containing the pT7-7/KIV-T6 plasmid and incubated at 37°C with agitation (250 r.p.m.) until the culture achieved an A600 nm of 0.5. At this point the expression of recombinant apo(a) KIV-T6 was induced by addition of 0.05 mM isopropyl-ß-D-thiogalactoside (IPTG) (Sigma) and the culture was incubated for an additional 4 h. During this time, A600 nm reached ~1.0 and ~1.9 for induced and non-induced cultures, respectively. Cells were harvested by centrifugation at 6000 g for 10 min at 4°C and the cell pellets were frozen at –70°C until use. Approximately 2 g of cells were obtained per liter of culture medium. Aliquots from induced and non-induced cells were taken, frozen at –20°C and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), followed by Coomassie Brilliant Blue staining in order to visualize protein bands. |
| IPTG |
| OD 0.5 =
600 |
| Not stated |
| None |
| Metal affinity chromatography |
| insoluble |
| Dilution |
| 20 mM Tris–HCl, 100 mM NaCl, 10 mM imidazole, final pH 8.0 |
| 100 mM Tris–HCl, 6 M guanidine–HCl (Sigma), 1 mM EDTA, 0.1 M DTT (Sigma), final pH 8.0 |
| 100 mM Tris–HCl, 0.4 M arginine (Sigma), 1 mM EDTA, 4 mM oxidized glutathione (Sigma), final pH 8.0 |
| Metal affinity chromatography |
| yes |
| 8.0 |
| 10.0 |
| n/a |
| 5 days |
| GSSG |
| 0.4 mM |
| Refolding of KIV-T6
This was performed as described (Buchner and Rudolph, 1991; LoGrasso et al., 1994). After exchange of elution buffer by ultrafiltration (Amicon membrane, YM3 = 3000 MW), recombinant protein from 1 l of E.coli culture was resuspended in 3 ml of buffer A [100 mM Tris–HCl, 6 M guanidine–HCl (Sigma), 1 mM EDTA, 0.1 M DTT (Sigma), final pH 8.0] and stirred (40 r.p.m.) at room temperature for 2 h. This solution was diluted with buffer B [100 mM Tris–HCl, 0.4 M arginine (Sigma), 1 mM EDTA, 4 mM oxidized glutathione (Sigma), final pH 8.0] to 100 ml and incubated at 10°C for 5 days. Samples were stored at –20°C until further use. |
| RP-HPLC,NMR analysis |
| None |
| L-Arginine |
| 0.4 M |
| 70% |
| n/a |
| n/a |