Yeast expression and characterization of SARS-CoV N protein.
The severe acute respiratory syndrome human coronavirus (SARS-CoV) nucleocapsid protein (N protein) is its most antigenic structural protein. The N protein gene has been cloned into a yeast expression vector pPIC9, transformed into Pichia pastoris strain GS115 and induced for expression by methanol. SDS-PAGE and Western blot showed that the N protein was expressed at a level of 3mg/ml of culture medium. Characterization by mass spectrometry, circular dichroism and fluorescence luminescence assays showed that the expressed N protein displayed a beta-sheet secondary structure in solution and it is stable in the pH range between 5.0 and 8.0. The P. pastoris-expressed N protein is believed to more closely resemble native SARS N protein than the bacterially expressed N protein.
Cao
Shishu
S
Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.
Wang
Haihe
H
Luhur
Arthur
A
Wong
Sek-Man
SM
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D000086462:Coronavirus Nucleocapsid Proteins; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D010843:Pichia / Q000378:metabolism*; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013696:Temperature
D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins