Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Nucleoprotein Severe acute respiratory syndrome coronavirus
Abbreviated Name	SARS-COV N
SCOP Family	Coronavirus nucleocapsid protein
Structure Notes
Organism	Human SARS coronavirus
UniProt Accession	P59595
SCOP Unique ID	n/a
Structure Solved
Class	Alpha+Beta
Molecularity	Dimer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	118
Molecular Weight	13319.0
Pi	9.77
Molecular Weight	13319.0
Disulphides	Unknown
Full Sequence	TKKSAAEASKKPRQKRTATKQYNVTQAFGRRGPEQTQGNFGDQDLIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYHGAIKLDDKDPQFKDNVILLNKHIDAYKTFP
Notes	n/a

Expression
Report	Chang CK, Sue SC, Yu TH, Hsieh CM, Tsai CK, Chiang YC, Lee SJ, Hsiao HH, Wu WJ, Chang CF, Huang TH. (2005) FEBS Letters, 579, 5663-5668
Project Aim	Undefined
Fusion	N-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)
Expression Temp	1.0
Expression Time	1
Expression Vector	pET6H
Expression Protocol	The fragments corresponding to residues 248–365 (NP248–365) and 281–365 (NP281–365) of SARS-CoV N proteins were expressed in Escherichia coli BL21(DE3) strain. Isotopically labeled protein samples were prepared by culturing the cells in standard M9 media, supplemented with 15NH4Cl (1 g/L) (For 15N-labeling) and/or u-13C-glucose (2 g/L) (For 13C-labeling) and appropriately labeled Isogro (0.5 g/L) (Isotec, OH, USA). Perdeuterated isotopically labeled protein samples were prepared by culturing the cells in the same media in D2O (80% D2O for samples used in filtered experiments) and supplemented with deuterated Isogro and glucose. Deuteration rates for all clones were on the order of 85% (65% for samples used in filtered experiments) as measured by mass spectrometry. The cells were broken with a microfluidizer and the protein purified through a Ni-NTA affinity column (Qiagen, CA, USA) in buffer (50 mM sodium phosphate, 150 mM NaCl, and pH 7.4) containing 7 M urea.
Method of Induction	Not Stated
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Microfluidizer
Lytic Agent	None
Pre-Refolding Purification	Metal affinity chromatography
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	n/a
Solubilization Buffer	50 mM sodium phosphate, 150 mM NaCl, and pH 7.4) containing 7 M urea
Refolding Buffer	50 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA, 0.01% NaN3, and pH 7.4
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	no
Refolding pH	7.4
Refolding Temperature	1.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	The protein was then allowed to refold by dialysis in liquid chromatography buffer (50 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA, 0.01% NaN3, and pH 7.4). Renatured protein was loaded onto an AKTA-EXPLORER fast performance liquid chromatography (FPLC) system equipped with a HiLoad 16/60 Superdex 75 column (Amersham Pharmacia Biotech, Sweden). Complete Protease Inhibitor cocktail (Roche, Germany) was added to the purified protein. Protein concentration was determined with the Bio-Rad Protein Assay kit as per instructions from the manufacturer (Bio-Rad, CA, USA). The correct molecular weight of the expressed protein was then confirmed by mass spectroscopy.
Refolding Assay	Mass spectrometry,Protein activity assay
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	Refolding temperature is not stated

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