Refold - Hepatitis C virus core protein

Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Hepatitis C virus core protein
Abbreviated Name	HCcAg
SCOP Family	Unknown
Structure Notes
Organism	Hepatitis C virus
UniProt Accession	Q9ELS8
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Dimer

Construct
Full Length	n
Domain	n/a
Chimera	n/a
Variants	Residues 1-169
Chain Length	177
Molecular Weight	19637.4
Pi	11.6
Molecular Weight	19637.4
Disulphides	0
Full Sequence	MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLLQHHHHHH
Notes	n/a

Expression
Report	Boulant, S., Vanbelle, C., Ebel, C., Penin, F., Lavergne, J. P (2005) J Virol, 79, 11353-11365
Project Aim	Structure-Function
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(SI)
Expression Temp	37.0
Expression Time	3h
Expression Vector	pT7-7
Expression Protocol	Cells were grown in LB medium without NaCl. They were then induced and left to incubated. Harvesting took place by centrifugation (5,500g, 10min, 4degC) and cells were resuspended in 25mM TrisHCl, pH 7.5, 5mM MgCl2, 1mM DTT, 1mM PMSF, 100U of benzonase/ml. Cells were then lyzed using a French pressure cell press at 1,200lb/in2. Inclusion bodies isolated by centrifugation(30,000g, 30min).
Method of Induction	NaCl
Cell Density at Induction	OD 600 = 0.7
Cell Disruption Method	French Press
Lytic Agent	None
Pre-Refolding Purification	Metal affinity chromatography
Solubility	insoluble

Refolding
Refolding Method	Column refolding: affinity immobilization
Wash Buffer	n/a
Solubilization Buffer	20 mM TrisHcl, pH 8.0, 500mM NaCl, 6M urea, 10mM beta-mercaptoethanl and 0.1% dodecylmaltoside.
Refolding Buffer	acetonitrile, 10% trifluoroacetic acid.
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	no
Refolding pH	7.5
Refolding Temperature	20.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	Beta-mercaptoethanol
Redox Agent Concentration	n/a
Refolding Protocol	Pellet was solubilized by addition of solubilisation buffer and sonication. The solution was then centrifuged(24,000g, 20min) and pellet subjected to a second urea extraction. Supernatants were then run over an equilibrated Ni-NTA-agarose column, which was washed with three volumes of solubilization buffer. Eluted proteins were subjected to reverse phase HPLC on a VYDAC C8 column using a linear gradient of refolding buffer. (0min, 25% acetonitrile; 0-5min 35% acetonitrile; 5-20min, 50% acetonitrile; 20-50min, 60% acetonitrile; 50-60min, 100% acetonitrile)
Refolding Assay	Far-UV Circular Dichroism
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a