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| Refolding Record [Created: 2008-12-15 Updated: 2008-12-15 ] |
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PROTEIN |
| Protein Name |
DASH complex subunit DAD1 |
| Abbreviated Name |
Dad1 |
| SCOP Family |
Unknown |
| Structure Notes |
Alternative name(s):
Outer kinetochore protein DAD1
DUO1 and DAM1-interacting protein 1 |
| Organism |
Saccharomyces cerevisiae |
| UniProt Accession |
Q12248 |
| SCOP Unique ID |
n/a |
| Structure Solved |
|
| Class |
Unknown |
| Molecularity |
Unknown |
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CONSTRUCT |
| Full Length |
y |
| Domain |
n/a |
| Chimera |
n/a |
| Variants |
n/a |
| Chain Length |
94 |
| Molecular Weight |
10515.7 |
| pI |
4.2 |
| Disulphides |
Unknown |
Pfam Domain(s) and
Number of Occurrences |
Pfam Entry
DASH_Dad1(1) |
| Domain Graphics |
 |
| Full Sequence |
MMASTSNDEEKLISTTDKYFIEQRNIVLQEINETMNSILNGLNGLNISLESSIAVGREFQSVSDLWKTLYDGLESLSDEAPIDEQPTLSQSKTK
BLAST against Refold database | BLAST against NCBI NR database
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| Notes |
n/a |
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EXPRESSION |
| Report |
Waldo J, Scherrer M. ( 2008) PLoS ONE, 3, 3888 |
| Project Aim |
Expression and charactrization |
| Fusion |
N-terminal hexahistidine tag |
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 |
pET15-DAD1 |
| Expression Protocol |
BL21(DE3) cells harboring pET15-DAD1 were inoculated into LB plus 100 µg/ml ampicillin and grown to saturation overnight. These cultures were then used to inoculate 1L cultures the following morning. The 1L cultures were grown to OD=0.4 at 37°C and protein expression was induced by the addition of 0.5 mM IPTG. Cells were harvested by centrifugation at 4°C after four hours growth and frozen at −80°C.
Cells were thawed in buffer A (50 mM Tris pH 8.0, .5 M NaCl) supplemented with lysozyme and sonicated. After centrifugation at 12,000×g for 30 minutes at 4°C, the pellet was resuspended in buffer A containing 8 M urea. The solution was centrifuged at 12,000×g for 30 minutes at 4°C. The supernatant was loaded onto a 10 ml column of chelating sepharose charged with nickel sulfate (GE Healthcare). The column was washed with Buffer A containing 8 M urea. Proteins were eluted by applying a linear gradient of immidazole (0 to .5 M) in the presence of 8 M urea. Fractions containing Dad1p were identified by SDS-PAGE. All chromatography steps were performed on an AKTA-FPLC (GE Healthcare). |
| Method of Induction |
IPTG |
| Cell Density (at induction) |
OD 0.4 = 600 |
| Cell Disruption Method |
Sonication |
| Lytic Agent |
Lysozyme |
| Pre-refolding Purification |
not specified |
| Solubility |
insoluble |
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REFOLDING |
| Refolding Method |
Dialysis |
| Wash Buffer |
50 mM Tris pH 8.0, .5 M NaCl |
| Solubilization Buffer |
8 M urea, 50 mM Tris pH 8.0, .5 M NaCl |
| Refolding Buffer |
1 M NaCl, and 50 mM Sodium Acetate buffer pH range of 5 to 6 |
| Pre-refolding Purification |
not specified |
| Tag Cleaved |
no |
| Refolding pH |
7.5 |
| Refolding Temperature |
4 |
| Protein Concentration |
n/a |
| Refolding Time |
overnight |
| Redox Agent |
None |
| Redox Agent Concentration |
n/a |
| Refolding Protocol |
One ml samples of the pooled fractions from the Nickel-sepharose column were placed into dialysis tubing and equilibrated overnight at 4°C against 1L of solutions buffered at various pHs. All solutions contained .1 M NaCl, and 50 mM buffer. For the pH range of 5 to 6, Sodium Acetate served as the buffer. For the pH range of 6 to 6.8, Pipes served as the buffer. For the pH range 6.5 to 7.5, Hepes was the buffer. For the pH range 7.5 to 9, Tris was the buffer. The pH of the buffer was measured after dialysis. Protein concentration was measured by the Bradford method (BioRad) before and after dialysis, following a 15-minute 13 K centrifugation step at 4°C. Additional purification steps: Following overnight dialysis into Buffer B (20 mM Tris, pH 7.5, 0.1 M NaCl, 2 mM EDTA), the sample was centrifuged and bound to a 1 ml monoQ column. The column was eluted with 20 mM Tris, pH 7.5, 0.6 M NaCl, 2 mM EDTA. Fractions containing Dad1p were pooled and concentrated with an Amicon Ultra 10.
Gel filtration analysis was conducted with a Superdex 200 (GE Healthcare) column equilibrated in buffer B. The column was calibrated with an equal volume of gel filtration standards (BioRad). |
| Assay |
Gel filtration chromatography |
| Chaperones |
None |
| Additives |
None |
| Additives Concentration |
n/a |
| Refolding Yield |
n/a |
| Purity |
n/a |
| Notes |
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