SARS CoV Nucleocapsid

Product Information

Product Description

SARS-CoV Nucleocapsid

Catalog Number
P1002

Description and Application
The coronavirus nucleocapsid protein is associated with viral RNA inside the virion. Sequence comparison of the N genes of five strains of the coronavirus mouse hepatitis virus suggests a three-domain structure for the nucleocapsid protein.

Sample Preparation
SUMO-Nucleocapsid fusion was expressed in E.coli in LB medium and purified using with Ni-NTA resin (Qiagen) affinity chromatography. After the fusion was cleaved by the SUMO Protease (LifeSensors), the SUMO tag and protease were subtracted from the nucleocapsid using MAC and the nucleocapsid was finally purified using Cation Exchange Chromatography with the Macro-Prep High S resin (BioRad) and size exclusion chromatography. The purified sample was extensively dialyzed against 10 mM ammonium bicarbonate, quantitiated using the Bradford color-reaction assay (BioRad reagents), measured spectrophotometrically at 595 mm, aliquoted, lyophilized, and stored at –20C.

Analysis of the Purified Sample



Purified SARS-CoV Nucleocapsid protein was run on a 15% SDS-acrylamide gel and stained by Coomassie Blue. Arrow highlights the Nucleocapsid protein band.

Lane M: Molecular weight markers
Lanes 1-3: 2 µg, 4 µg, and 6 µg of the protein, respectively.

Sample Storage and Stability:
Lyophilized protein samples are stable for several weeks at 4C. No protein breakdown was observed even at 37C for six days. The dried protein samples can be shipped at room temperature by mail. However, we recommend –20C or -80C storage for longevity of the samples.

References
Zuo, X., et al. (2005). “Expression and purification of SARS Coronavirus Proteins using SUMO Fusions.” Protein Expres Purif (In Press).

Zuo, Xun., et al. (2005). “Enhanced expression and purification of membrane proteins by SUMO fusion in Escherichia coli.” J Struct Funct Genomics (In Press).

Malakhov, M. P., et al. (2004). “SUMO fusion and SUMO-specific protease for efficient expression and purification of proteins.” J Struct Funct Genomics 5(1-2): 75-86.

Butt, T. R. et al. (2005). “SUMO fusion technology for difficult-to-express proteins.” Protein Expres Purif (In Press).

Marra, M.A., et al. (2003). "The genome sequence of the SARS-associated coronavirus." Science 300(5624): 1399-1404.

M. Surjit, et al. (2004). "The nucleocapsid protein of the SARS coronavirus is capable of self-association through a C-terminal 209 amino acid interaction domain." Biochem. Biophys. Res. Commun. 317(4): 1030-1036.

Lin Y. et al. (2003). "Identification an epitope of SARS-coronavirus nucleocapsid protein." Cell Research 13(3): 141-145.

Narayanan, K., et al. (2000). "Characterization of the coronavirus M protein and nucleocapsid interaction in infected cells." J. Virol. 74(17): 8127-8134.

T.G. Ksiazek, et. al. (2003). "A novel coronavirus associated with severe acute respiratory syndrome." N. Engl. J. Med. 348(20): 1953-1966.

P.A. Rota, et al. (2003). "Characterization of a novel coronavirus associated with severe acute respiratory syndrome." Science 300(5624): 1394-1399.

S. Muller, et al. (2001). "SUMO, ubiquitin's mysterious cousin." Nat. Rev. Mol. Cell Biol. 2(3): 202-210.

F. Melchior (2000). "SUMO--nonclassical ubiquitin." Annu. Rev. Cell Dev. Biol. 16: 591-626.