| Farina A, Faiola F, Martinez E.
(2004)
Protein Expression and Purification,
34,
215-222 |
| Undefined |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3)CodonPlus |
| 30.0 |
| 3 h |
| pRSET-6His-Myc |
| Expression of recombinant c-Myc and Max in Escherichia coli
pRSET-6His-Myc expression vector was transformed into BL21-CodonPlus(DE3)-RP competent cells (Stratagene). pET-His-Max was transformed into BL21(DE3)pLysS competent cells (Stratagene). Bacteria were grown in LB broth plus 100 μg/ml ampicillin at 30 °C and protein expression was induced with 0.5 mM IPTG at 0.3–0.4 OD600 nm. Induction was performed for 3 h at 30 °C. Bacteria were collected and washed with cold washing buffer (10 mM Tris–HCl, pH 7.9, at 4 °C, 100 mM NaCl, and 1 mM EDTA), and cell pellets were either used directly for purification or stored frozen at −20 °C until further use. |
| IPTG |
| OD 0.3-0.4 =
600 |
| Sonication |
| None |
| Metal affinity chromatography |
| insoluble |
| Dialysis |
| S-buffer containing 5 mM imidazole, 3 times with 1 ml BC500 containing 7 M urea and 5 mM imidazole |
| S-buffer (10 mM Hepes, pH 7.9, 6 M guanidine–HCl, and 5 mM of 2-mercaptoethanol) |
| 7 successive steps of 2 h each against BC500 (as above, but with 0.1% NP-40) containing 4 M urea (BC500—4 M urea), BC500—2 M urea, BC500—1 M urea, BC500—0.5 M urea, BC500, and twice with BC100 |
| Metal affinity chromatography |
| no |
| 0.0 |
| 25.0 |
| n/a |
| 14 h |
| None |
| n/a |
| To co-renature a significant amount of Myc:Max heterodimers over Max:Max homodimers and to minimize formation of soluble Myc homodimers, a 3:1 molar ratio of Myc to Max was used during a slow stepwise dialysis. 1.5 μg of purified Max protein was mixed with 15 μg purified Myc protein in 150 μl BC100 containing 7 M urea and dialyzed in “Mini Dialysis Tubes” (nominal molecular weight cutoff at 8 kDa, Amersham) in 7 successive steps of 2 h each against BC500 (as above, but with 0.1% NP-40) containing 4 M urea (BC500—4 M urea), BC500—2 M urea, BC500—1 M urea, BC500—0.5 M urea, BC500, and twice with BC100. First four dialysis steps were done at room temperature, while the last three were done at 4 °C. After the last dialysis step, the dialyzed proteins were centrifuged at 13,000 rpm for 1 h at 4 °C in an Eppendorf microfuge to remove insoluble aggregates. An aliquot (1 μl) was analyzed by SDS–PAGE and Coomassie staining (Fig. 1B, lane 1) and bovine serum albumin (BSA, 500 ng/μl final concentration) was added to the remaining Myc:Max preparation and stored in small aliquots at −80 °C. No noticeable decrease in DNA-binding activity was observed after up to 10 freeze–thaw cycles. Typically 100% of Max and about 50–60% of total Myc were recovered in the soluble fraction after dialysis. When Myc was dialyzed alone (in the absence of Max), only about 16% of total Myc was recovered as soluble protein. Thus, after dialysis we estimate that more than 50% of renatured Myc is in a soluble heteromeric complex with Max. |
| Electrophoretic mobility-shift assays |
| None |
| None |
| n/a |
| n/a |
| n/a |
| In the refolding protocol the MAX protein use as a pseudo chaperon. |