Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Diisopropylfluorophosphatase
Abbreviated Name	DFPase
SCOP Family	Diisopropylfluorophosphatase (phosphotriesterase, DFP)
Structure Notes
Organism	Loligo vulgaris
UniProt Accession	Q7SIG4
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Monomer

Construct
Full Length	n
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	176
Molecular Weight	20115.8
Pi	7.15066
Molecular Weight	20115.8
Disulphides	0
Full Sequence	VA PADYTRSMQE KFGSIYCFTT DGQMIQVDTA FQFPNGIAVR HMNDGRPYQL IVAETPTKKL WSYDIKGPAK IENKKVWGHI PGTHEGGADG MDFDEDNNLL VANWGSSHIE VFGPDGGQPK MRIRCPFEKP SNLHFKPQTK TIFVTEHENN AVWKFEWQRN GKKQYCETLK FGIF
Notes	n/a

Expression
Report	Hartleib J, Ruterjans H. (2001) Protein Expression and Purification, 21, 210-219
Project Aim	Structural Studies
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21
Expression Temp	37.0
Expression Time	4h
Expression Vector	pTHis, pET24d
Expression Protocol	Overnight colonies harbouring the relevant expression plasmids were used to inoculate 500ml LB media supplemented with 50 micrograms/ml kanamycin. Cells were grown at 37degC until OD600 reached 0.8, expression was induced with 1mM IPTG and cells were incubated a further 4 hours. Cells were then harvested.
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.8
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	n/a
Solubilization Buffer	50mM TrisHCl, 6M urea, pH 8.5
Refolding Buffer	various conditions (see paper)
Pre-Refolding Purification	None
Tag Cleaved	yes
Refolding pH	6.5
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	12h
Redox Agent	None
Redox Agent Concentration	n/a,n/a
Refolding Protocol	Cell pellets were resuspended in 5ml sonication buffer(50mM Tris/HCl, 20%(w/v) sucrose, pH 8.0). The suspensio nwas then frozen and thawed on ice, followed by lysis by sonicatin. The inclusion bodies were collected by centrifugation (20min x 20800g, 4degC) and resuspended in solubilization buffer. The suspension was swirled on ice for 1-2h to enable full solubilization, then the mixture was removed by centrifugation (20min x 20800g, 4degC) to remove any remaining insoluble material. The protein concentration of the supernatant was then measured using an aborbance coefficient of 31ml/mg/cm at 205nm. For refolding, the protein concentration was adjusted to 0.1-1mg/ml. The protein fragments were dialized twice against 2.5L refolding buffer for 12 hours. Following dialysis the protein solutions were centrifuged (10min x 20800g, 4degC) and the supernatant analyzed for enzyme activity.
Refolding Assay	Far-UV Circular Dichroism,Enzyme activity,SDS-PAGE
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	- full length protein expressed and purified solubly - proteolytic fragments NF (aa1-148) and CF (149-314) were expressed insolubly and purified from inclusion bodies - several different refolding buffer conditions investigated (see paper for more details)

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