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| Refolding Record [Created: 2008-12-08 Updated: 2008-12-08 ] |
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PROTEIN |
| Protein Name |
Activating transcription factor 5 |
| Abbreviated Name |
ATF-5 |
| SCOP Family |
Unknown |
| Structure Notes |
Also known as : Cyclic AMP-dependent transcription factor ATF-5 |
| Organism |
Human |
| UniProt Accession |
Q9Y2D1 |
| SCOP Unique ID |
n/a |
| Structure Solved |
homologue only |
| Class |
Unknown |
| Molecularity |
Unknown |
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CONSTRUCT |
| Full Length |
n |
| Domain |
bZIP domain |
| Chimera |
n/a |
| Variants |
n/a |
| Chain Length |
16 |
| Molecular Weight |
1716.9 |
| pI |
4.49 |
| Disulphides |
Unknown |
Pfam Domain(s) and
Number of Occurrences |
Pfam Entry
Entry does not contain any Pfam domains |
| Domain Graphics |
Graphics not available for entry |
| Full Sequence |
LEGECQGLEARNREL
BLAST against Refold database | BLAST against NCBI NR database
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| Notes |
n/a |
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EXPRESSION |
| Report |
Ciaccio et al., ( 2008) Protein Expression and Purification, 62, 235-43 |
| Project Aim |
Protein refolding |
| Fusion |
N-terminal GST |
Protein Expression and
Production |
Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host |
E coli |
| Expression Strain |
BL21 (DE3) |
| Expression Temperature |
37 |
| Expression Time |
4 h |
| Expression Vector |
pET-42b-ATF5 bZIP |
| Expression Protocol |
BL21 (DE3) E. coli (Novagen) were transformed with the pET-42b-ATF5 bZIP plasmid using the standard heat shock protocol. Transformed colonies were selected and grown overnight at 37 °C on LB agar plates containing 30 μg/mL kanamycin. Colonies were selected and used to inoculate 5 mL LB containing 0.4% glucose, 1 mM MgSO4, and 30 μg/mL kanamycin. Cultures were grown with shaking overnight at 37 °C. Two starter cultures were used to inoculate one 500 mL culture containing minimal media consisting of deionized water supplemented with 0.1% ammonium chloride, 1% glucose, 10 mM MgCl, 40 μg/mL thiamine HCl, 30 μg/mL kanamycin plus additional salts (100 mM KH2PO4, 57 mM K2HPO4, 63 mM Na2HPO4, 14 mM K2SO4) and trace minerals (200 μM CaCl2·H2O, 100 μM FeSO4·H2O, 50 μM MnCl2·6H2O, 15 μM CoCl2·6H20, 10 μM ZnSO4·7H2O, 9 μM CuCl2·2H2O, 1.5 μM H3BO4, 1.2 μM (NH4)Mo7O24·4H2O, 65 μM Na2EDTA). Isotopic labeling was accomplished via substitution with 15N-labeled ammonium chloride (Spectra Isotopes). Cultures were grown to an OD550 of 0.6–0.8 before inducing protein expression with a final concentration of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Cells were incubated with shaking at 37 °C for 4 additional hours post-induction, harvested by centrifugation at 3000g and stored at −80 °C. |
| Method of Induction |
IPTG |
| Cell Density (at induction) |
OD 0.6-0.8 = 550 |
| Cell Disruption Method |
French Press |
| Lytic Agent |
None |
| Pre-refolding Purification |
not specified |
| Solubility |
insoluble |
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REFOLDING |
| Refolding Method |
Dilution |
| Wash Buffer |
n/a |
| Solubilization Buffer |
50 mM sodium phosphate, pH 6.5 containing 100 mM NaCl, 6 M guanidine HCl, and 5 mM DTT |
| Refolding Buffer |
50 mM sodium phosphate, pH 6.5 containing 100 mM NaCl and 5 mM DTT |
| Pre-refolding Purification |
not specified |
| Tag Cleaved |
no |
| Refolding pH |
6.5 |
| Refolding Temperature |
4 |
| Protein Concentration |
n/a |
| Refolding Time |
overnight |
| Redox Agent |
DTT |
| Redox Agent Concentration |
5 mM |
| Refolding Protocol |
Cell pellets were thawed, resuspended in PBS, pH 7.3 containing 5 mM DTT and lysed as described above. The insoluble pellet was resuspended by vortexing it in 4 mL of 50 mM sodium phosphate, pH 6.5 containing 100 mM NaCl, 6 M guanidine HCl, and 5 mM DTT. The solution was incubated at room temperature for 3 h to permit denaturation to occur before separating the soluble and insoluble material by centrifugation for 1 h at 21,000g, and 4 °C. The solubilized protein was refolded by dilution into 40 mL of 50 mM sodium phosphate, pH 6.5 containing 100 mM NaCl and 5 mM DTT [18]. Insoluble components were pelleted by centrifugation for 1 h at 21,000g and 4 °C. The supernatant containing refolded ATF5 was dialyzed using 1000 MWCO dialysis tubing with a cellulose membrane (Spectra) overnight at 4 °C into 2 L of phosphate buffer (50 mM sodium phosphate, pH 6.5, 100 mM NaCl, 5 mM DTT) to remove the guanidinium. The ratio of protein to dialysis solution was 0.04 L protein: 2 L dialysis buffer, which results in a 50-fold dilution of guanidinium to a final concentration of 12 mM. Any precipitation generated during dialysis was removed by centrifugation for 1 h at 21,000g and 4 °C. The supernatant containing soluble protein was collected and concentrated using a 15 mL 3500 MWCO Millipore ultrafilter with a Biomax membrane and exchanged into the same buffer solution (50 mM sodium phosphate, pH 6.5, 100 mM NaCl) without DTT. The solution was concentrated to approximately 1 mL and then exchanged into 20-fold excess buffer to further remove guanidinium, reducing the final concentration to less than 1 mM. Sample purity was assessed by SDS–PAGE analysis. |
| Assay |
NMR analysis, Activity assay, Mass spectrometry |
| Chaperones |
None |
| Additives |
None |
| Additives Concentration |
n/a |
| Refolding Yield |
n/a |
| Purity |
n/a |
| Notes |
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