Expression, purification and sublocalization of SARS-CoV nucleocapsid protein in insect cells.
The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is a major viral protein recognized by acute and early convalescent sera from SARS patients. To facilitate the studies on the function and structure of the N protein, this report describe the expression and purification of recombinant SARS-CoV N protein using the baculovirus expression system. Recombinant hexa-histidine-tagged N protein with a molecular mass of 47 kD was produced in insect cells. Recombinant N protein was purified to near homogeneity by Ni2+-NTA affinity chromatography. In addition, we examined the subcellular localization of the N protein by confocal microscopy in Trichoplusia ni BT1 Tn 5B1-4 cells infected with recombinant baculovirus. The N protein was found localized in the cytoplasm as well as in the nucleolus. The purified recombinant N protein can be used in further functional study of SARS-CoV.
Ren
Ai-Xia
AX
Institute of Biochemistry and Biotechnology, College of Life Science, Zhejiang University, Hangzhou 310029, China.
Xie
You-Hua
YH
Kong
Yu-Ying
YY
Yang
Guan-Zhen
GZ
Zhang
Yao-Zhou
YZ
Wang
Yuan
Y
Wu
Xiang-Fu
XF
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D000086462:Coronavirus Nucleocapsid Proteins; D003593:Cytoplasm / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007313:Insecta; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D019590:Nucleocapsid Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000378:metabolism*
D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins