Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Cell division protein ftsH
Abbreviated Name	ftsH
SCOP Family	Extended AAA-ATPase domain
Structure Notes
Organism	Escherichia coli
UniProt Accession	P0AAI3
SCOP Unique ID	n/a
Structure Solved
Class	Alpha/Beta
Molecularity	Hexamer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	644
Molecular Weight	70708.1
Pi	5.91
Molecular Weight	70708.1
Disulphides	0
Full Sequence	MAKNLILWLVIAVVLMSVFQSFGPSESNGRKVDYSTFLQEVNNDQVREARINGREINVTK KDSNRYTTYIPVQDPKLLDNLLTKNVKVVGEPPEEPSLLASIFISWFPMLLLIGVWIFFM RQMQGGGGKGAMSFGKSKARMLTEDQIKTTFADVAGCDEAKEEVAELVEYLREPSRFQKL GGKIPKGVLMVGPPGTGKTLLAKAIAGEAKVPFFTISGSDFVEMFVGVGASRVRDMFEQA KKAAPCIIFIDEIDAVGRQRGAGLGGGHDEREQTLNQMLVEMDGFEGNEGIIVIAATNRP DVLDPALLRPGRFDRQVVVGLPDVRGREQILKVHMRRVPLAPDIDAAIIARGTPGFSGAD LANLVNEAALFAARGNKRVVSMVEFEKAKDKIMMGAERRSMVMTEAQKESTAYHEAGHAI IGRLVPEHDPVHKVTIIPRGRALGVTFFLPEGDAISASRQKLESQISTLYGGRLAEEIIY GPEHVSTGASNDIKVATNLARNMVTQWGFSEKLGPLLYAEEEGEVFLGRSVAKAKHMSDE TARIIDQEVKALIERNYNRARQLLTDNMDILHAMKDALMKYETIDAPQIDDLMARRDVRP PAGWEEPGASNNSGDNGSPKAPRPVDEPRTPNPGNTMSEQLGDK
Notes	proposed molecularity is hexameric

Expression
Report	Bruckner, R. C., Gunyuzlul, P. L., Stein, R. L. (2003) Biochemistry, 42, 10843-1852
Project Aim	Biophysical Studies
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)
Expression Temp	28.0
Expression Time	3h
Expression Vector	pET-11a
Expression Protocol	Cells grown in 750ml of LB media containing 100microg/l ampicillin and induced. After incubation cells were harvested by centrifugation(10,000g, 20 min), resuspended in 40ml of BPER containing protease inhibitors, and frozen. Cells were lysed on thawing by the addition of 280 microL of buffer A (0.71M MgCl2, 0.14M DTT,14.3mM ZnCl2 + 10 000 units DNase I and 8mg, lysozyme. This mixture was then incubated for 45min at 22degC, prior to centrifugation to obtain inclusion bodies(47,000g, 30min).
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Freeze-thaw
Lytic Agent	Lysozyme
Pre-Refolding Purification	Washing inclusion body
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	A. 50mM TrisHCl, pH 8.0, 1%(v/v) NP-40, 1mM DTT, 5mM MgCl2, 100microM ZnCl2 and protease inhibitor cocktail II. B. 50mM TrisHCl, pH 8.0, 0.1%(v/v) NP-40, 1mM DTT, 5mM MgCl2, 100microM ZnCl2 and
Solubilization Buffer	Novagen Solubilization Buffer, 5mM MgCl2, 100microM ZnCl2, 1 mM DTT, 1mM ATP, 0.6% N-laurylsarcosine
Refolding Buffer	4L of 25mM TrisHCl, pH 8.5, 5mM MgCl2, 100microM ZnCl2, 01% NP40, 1mM DTT
Pre-Refolding Purification	Washing inclusion body
Tag Cleaved	no tag
Refolding pH	8.5
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	30min
Redox Agent	DTT
Redox Agent Concentration	1mM
Refolding Protocol	Pellets were then resuspended in 40mL of wash buffer A, with gentle stirring at 4degC for 30min. This mixture was then centrifuged(47,000g, 30min), before resulting pellets were then resuspended in 40ml of Wash Buffer B with 0.1% NP-40 and incubated for 30min. This mixture was then separated into equal volumes and pelleted. Pellets were resuspended in 9.8 ml solubilization buffer, with gentle stirring for 30min at 22degC. Insoluble material was removed by centrifugation(16,000g, 10min). 25ml of pre-refolding buffer (50mM Tris-HCL, pH 8.0, 0.5% NP40, 5mM MgCl2, 100microM ZnCl2, 1 mM DTT, 1 mM ATP) was then added at 1 ml at a time over 30min. The mixture was allowed to stand on ice for 30min prior to overnight dialysis against refolding buffer at 4degC.
Refolding Assay	Enzyme activity
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

Back to refolding records...