Refold - Type II DNA topoisomerase VI

Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Type II DNA topoisomerase VI
Abbreviated Name	TopoVI
SCOP Family	DNA topoisomerase IV, alpha subunit
Structure Notes
Organism	Sulfolobus shibatae
UniProt Accession	O05208
SCOP Unique ID	n/a
Structure Solved
Class	Multi-domain proteins (alpha and beta)
Molecularity	Tetramer

Construct
Full Length	n
Domain	A subunit
Chimera	n/a
Variants	n/a
Chain Length	427
Molecular Weight	49268.5
Pi	6.56
Molecular Weight	49268.5
Disulphides	0
Full Sequence	MSSEFISKVDKEARRKAANILRDKFLNLVEQLKKGEPLVMEIPMRTLSNAIYDEKRKLLL LGEKKLRRNFLDLNEAKRFMQTVLMASIIYDALVSDEYPTIRDLYYRGKHSLLLKSIEGN KIVSEENTWDEQKESDSVIVDIEVFTSLLREEMLILSKEKGKVVGNLRIRSGNDVIDLSK TGHGAYAIEPTPDLIDFIDVDAEFVLVVEKDAVFQQLHRAGFWKQYKSILITSAGQPDRA TRRFVRRLNEELKLPVYILTDADPYGWYIFSVFRIGSISLSYESERLATPDAKFLGVSMG DIFGNSRKKPYLSEAERKNYIIKAKDADIKRAEEIKNYEWFKTKAWQEEINTFLQRKAKL EIEAMASKGLKFLAFQYIPEKITNKDYIADPNSSSVDKLAAALEKRASQPELAPEDPEDVEHHHHHH
Notes	n/a

Expression
Report	Buhler, C., Gadelle, D., Forterre, P., Wang, J. C., Bergerat, A. (1998) Nucleic Acids Research, 26, 5157-5162
Project Aim	Assembly
Fusion	C-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)pLysS
Expression Temp	37.0
Expression Time	2
Expression Vector	pET-25b
Expression Protocol	Cells were grown in 2L of LB medium (100microg/ml ampicillin and 50microg/ml chloramphenicol). After induction the cells were harvested and resuspended in 25ml extraction buffer (50mM Tris, pH 7.5, 300mM NaCl, 1mM EDTA, 1% Triton X-100 and 0.5 mg/ml lysozyme) and incubated for 30min at 4degC. Cell paste was blended in a rotarty homogenizer and inclusion bodies isolated by the addition of 75ml TE (10mM TrisHCl, pH 8.0, 0.1 mM EDTA) and 50ml of SciI buffer (1%NP-40, 0.5% desoxycholate, 0.5% Triton X-100, 100mM NaCL, 10mM TrisHCl, pH 7.5) prior to centrifugation.
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.9
Cell Disruption Method	High pressure homogenization
Lytic Agent	Lysozyme
Pre-Refolding Purification	Washing inclusion body
Solubility	not stated

Refolding
Refolding Method	Dilution
Wash Buffer	A. SciI buffer (1%NP-40, 0.5% desoxycholate, 0.5% Triton X-100, 100mM NaCL, 10mM TrisHCl, pH 7.5) B. TE Buffer (10mM TrisHCl, pH 8.0, 0.1 mM EDTA)
Solubilization Buffer	6M guanidinium chloride
Refolding Buffer	50mM Tris, pH 7.5, 1mM EDTA, 20% ammonium sulfate
Pre-Refolding Purification	Washing inclusion body
Tag Cleaved	no
Refolding pH	7.5
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Inclusion bodies were washed 3x with 50ml of washing buffer A and 2x with 20ml washing buffer B. Between each wash the pellet was centrifuged(27,000g, 10min). The pellet was then solubilized and incubated for 2 hours at 40degC. Renaturation was done by a rapid 60x dilution into refolding buffer. This was then incubated for 2h at 40degC before centrifugation(40000, 30min). Supernatant was then loaded onto a phenyl-Sepharose column pre-equilbrated with refolding buffer and washed with 4 volumes worth of refolding buffer, 2 volumes worth of Buffer A (50mM Tris, pH 7.5, 1 mM EDTA, 250mM NaCl) and 2 volumes of buffer B, (50mM Tris, pH 7.5, 1mM EDTA, 50% ethylene glycol). Protein was then eluted with buffer C (50mM Tris, pH 7.5, 1mM EDTA, 70% ehtylene glycol)
Refolding Assay	ATP hydrolysis,Complex Assembly
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a