Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Tumor suppressor p16
Abbreviated Name	p16
SCOP Family	Unknown
Structure Notes
Organism	Human
UniProt Accession	P42771
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Monomer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	175
Molecular Weight	18690.2
Pi	6.52
Molecular Weight	18690.2
Disulphides	0
Full Sequence	MEPSADWLATAAARGRVEEVRALLEAGALPNAPNSYGRRPIQVMMMGSARVAELLLLHGAEPNCADPATLTRPVHDAAREGFLDTLVVLHRAGARLDVRDAWGRLPVDLAEELGHRDVARYLRAAAGGTRGSNHARIDAAEGPSDIPDSEPELRRQACACGRTRAPPPPPLRSGC
Notes	n/a

Expression
Report	Boice, J., Fairman, R. (1996) Protein Science, 5, 1776-1784
Project Aim	Structural Studies
Fusion	C-terminal sequence
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)
Expression Temp	37.0
Expression Time	4h
Expression Vector	pET-29a
Expression Protocol	1L M9 minimal media containing casamino acids, trace minerals, glycose and 25microg/ml kanamycin was innoculated with 20ml overnight culture. After induction cells were harvested by centrifugation and resuspended in 30ml buffer A (20mM TrisHCl, pH 7.5, 1mM EDTA, 5mM DTT, and protease inhibitors). Cells were then lyzed by one freeze/thaw cycle, followed by French Press and inclusion bodies were obtained after centrifugation(50,000g, 20min).
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 1.0
Cell Disruption Method	Freeze/Thaw+French Press
Lytic Agent	None
Pre-Refolding Purification	Ion-exchange chromatography
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	n/a
Solubilization Buffer	8M Urea, 20mM TrisHCl, pH 7.5, 1mM EDTA, 5mM DTT, and protease inhibitors
Refolding Buffer	1M Urea, 20mM TrisHCl, pH 7.5, 1mM EDTA, 5mM DTT, and protease inhibitors, pH 8.4
Pre-Refolding Purification	Ion-exchange chromatography
Tag Cleaved	no
Refolding pH	8.4
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	15h
Redox Agent	DTT
Redox Agent Concentration	5mM
Refolding Protocol	Gelatinous top layer of pellet was removed and the remaining pellet was resuspended in 30ml buffer A. This solution was then centrifuged before soubilization in 30ml solubilization buffer followed by incubation on a rotator for 1h at room temp and then overnight at 4degC. The pH was adjusted to 8.8 and fractionated on a 20ml bed of Q-sepharose anion exchange resin equilibrated with solubilization buffer,(pH 8.8). Protein was eluted using a linear gradient of 0-300mM NaCl in solubilisation buffer. S-Tag-p16 eluted at 100mM and fractions were pooled before pH was adjusted to 4.8, and overnight dialysis against 40 volumes of buffer B (50mM Na acetate, pH 4.8, 1mM EDTA, 5mM DTT, 8M urea) was preformed. This was then passed over S-sepharose cation exchange resin, equilibrated with buffer B and eluted with a linear gradient of 0-250mM NaCl. Urea solubilized S-tag-o16 was refolded by dilution in refolding buffer.
Refolding Assay	Structure Determination
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	35microg/ml
Purity	n/a
Notes	n/a

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