Refold - Cauliflour mosaic virus capsid protein

Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Cauliflour mosaic virus capsid protein
Abbreviated Name	CaMV
SCOP Family	Unknown
Structure Notes
Organism	Cauliflower Mosaic Virus
UniProt Accession	Q83168
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	497
Molecular Weight	56705.0
Pi	5.27
Molecular Weight	56705.0
Disulphides	Unknown
Full Sequence	MAESILDRTINRFWYNLGEDCLSESQFDLMIRLMEESLDGDQIIDLTSLPSDNLQVEPVM TTTDDSISEEESEFLLAIGETSEDESDSGEEPEFEQVRMDRTGGTEIPKEEDGEPSRYNE RKRKTTEGRYFPTQPKTIPGQKQTSMGMLNIDCQTNRRTLIDDWAAEIGLIVKTNREDYL DPETILLLMEHKTSGIPKELIRNTRWNRTTGDIIEQVIDAMYTMFLGLNYSDNKVAEKID EQEKAKIRMTKLQLCDICYLEEFTCDYEKNMYKTELADFPGYINQYLSKIPIIGEKALTR FRHEANGTSIYSLGFAAKIVKEELSKICDLSKKQKKLKKFNKKCCSIGEASAEYGCKKTS TKKYHKKRYKKKYKVYKPYKKKKKFRSGKYFKPKEKKGSKQKYCPKGKKDCRCWICNIEG HYANECPNRQSSEKAHILQQAEKLGLQPIEEPYEGVQEVFILEYKEEEEETSTEESDDES STSEDSDSD
Notes	full lenght wildtype protein and a series of delition derivatives were refolded with the same method though different buffers

Expression
Report	Chapdelaine Y and Hohn T (1998) Virus Genes, 17, 139-150
Project Aim	Assembly
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	37.0
Expression Time	2 h
Expression Vector	pET3d
Expression Protocol	Constructs were introduced into Escherichia coli strain BL21 (DE3) by transformation. Liquid cultures were started from several single bacterial colonies in LB medium supplemented with 0.4% glucose and containing 50 mg/ml ampicillin. The cultures were grown to A600 ? 0:6 ÿ 1:0 and expression was induced with 10 mg/ml isoproyl-û-D-thiogalactopyranosid (IPTG) for 2 h at 37 C (22). Cell suspensions were cooled on ice and spun at 5000 g for 15 min. The bacterial pellets were washed and resuspended in lysis buffer (20mM Tris-HCl, pH 7.5, 10mM EDTA, 1mM dithiothreitol (DTT) and 1mM phenylmethylsulfonyl ¯uoride (PMSF, (23)) before being treated with lysozyme (2 mg/ml of culture) for 30 min on ice, and frozen on dry ice. After thawing, lysates were sonicated and spun at 10,000 g for 10 min.
Method of Induction	IPTG
Cell Density at Induction	OD 0.6-1.0 = 600
Cell Disruption Method	Freeze/Thaw+Sonication
Lytic Agent	Lysozyme
Pre-Refolding Purification	Washing inclusion body
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	20mM Tris-HCl, pH 7.5, 10mM EDTA, 1mM DTT ,1 mM phenylmethylsulfonyl fluoride (PMSF)
Solubilization Buffer	8M urea
Refolding Buffer	20mM Tris-HCl, pH 7.5, 10mM EDTA, 1mM DTT , 0.1mM PMSF , 0.1% Tween-20
Pre-Refolding Purification	Washing inclusion body
Tag Cleaved	no tag
Refolding pH	7.5
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	overnight
Redox Agent	DTT
Redox Agent Concentration	n/a,1 mM,n/a,n/a,n/a,n/a,n/a,n/a,n/a,1 mM,n/a,n/a,n/a,n/a,n/a,1 mM
Refolding Protocol	Inclusion body pellets were washed, resolubilized in 8M urea and renatured by dialysis overnight at 4 C against the lysis buffer (containing 0.1mM PMSF and 0.1% Tween-20) OR PBS; 10 mM Na2HPO4 + KH2Po4, 140 mM NaCl, 5.4 mM KCl. After dialysis, samples were clari®ed by centrifugation (10,000 g for 10 min). The supernatant contained between 0.5 to 2 mg/ml of recombinant pIV. Renatured proteins (200 mg) were loaded on the top of 10±60% sucrose gradients and centrifuged at 40,000 rpm in a SW40 rotor for 4 h at 20 C.
Refolding Assay	Unspecified,SDS-PAGE
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	Includes constructs, pIV1-489, 1-454, 1-362, 1-265, 77-489, 77-480, 77-411, 77-362, 77-265, 99-454, 99-265, 99-255. A second buffer was used for refolding pIV77-332, and has another entry in database - PBS; 10 mM Na2HPO4 + KH2PO4, pH 7.3, 140 mM NaCl, 5.4 mM KCl