Refolding Record:

Protein See all refolding records for this protein...
Protein Name	DASH complex subunit DAD1
Abbreviated Name	Dad1
SCOP Family	Unknown
Structure Notes
Organism	Saccharomyces cerevisiae
UniProt Accession	Q12248
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	94
Molecular Weight	10515.7
Pi	4.2
Molecular Weight	10515.7
Disulphides	Unknown
Full Sequence	MMASTSNDEEKLISTTDKYFIEQRNIVLQEINETMNSILNGLNGLNISLESSIAVGREFQSVSDLWKTLYDGLESLSDEAPIDEQPTLSQSKTK
Notes	n/a

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 Temp	37.0
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

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	4.0
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).
Refolding Assay	Gel filtration chromatography
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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