Expression, purification and characterization of recombinant severe acute respiratory syndrome coronavirus non-structural protein 1.
The coronavirus (CoV) responsible for severe acute respiratory syndrome (SARS), SARS-CoV, encodes two large polyproteins (pp1a and pp1ab) that are processed by two viral proteases to yield mature non-structural proteins (nsps). Many of these nsps have essential roles in viral replication, but several have no assigned function and possess amino acid sequences that are unique to the CoV family. One such protein is SARS-CoV nsp1, which is processed from the N-terminus of both pp1a and pp1ab. The mature SARS-CoV protein is present in cells several hours post-infection and co-localizes to the viral replication complex, but its function in the viral life cycle remains unknown. Furthermore, nsp1 sequences are highly divergent across the CoV family, and it has been suggested that this is due to nsp1 possessing a function specific to viral interactions with its host cell or acting as a host specific virulence factor. In order to initiate structural and biophysical studies of SARS-CoV nsp1, a recombinant expression system and a purification protocol have been developed, yielding milligram quantities of highly purified SARS-CoV nsp1. The purified protein was characterized using circular dichroism, size exclusion chromatography, and multi-angle light scattering.
Brucz
Kimberly
K
Hauptman-Woodward Medical Research Institute, 700 Ellicott Street, Buffalo, NY 14203, USA.
Miknis
Zachary J
ZJ
Schultz
L Wayne
LW
Umland
Timothy C
TC
D002850:Chromatography, Gel; D002942:Circular Dichroism; D015870:Gene Expression; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000737:chemistry* / Q000235:genetics; D017361:Viral Nonstructural Proteins
D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D010447:Peptide Hydrolases