Expression, purification and characterization of recombinant severe acute respiratory syndrome coronavirus non-structural protein 1

Abstract

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 colocalizes 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 SARSCoV 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.The Severe Acute Respiratory Syndrome (SARS) outbreak of 2002-2003, followed by a much smaller outbreak in 2004, caused over 8000 illnesses and nearly 800 deaths (World Health Organization; http://www.who.int/csr/sars/country/table2004_04_21/en/index.html). The infectious agent responsible for this disease was quickly identified as a new member of the coronavirus (CoV) family, SARS-coronavirus (SARS-CoV) [1][2][3], most closely related to the group 2 CoVs [4]. This newly emerged virus prompted a renewed interest in CoV research. Prior to the SARS outbreak, only two CoVs (HCoV-229E and HCoV-OC43) were known to infect humans [5]. These two CoVs have been estimated to cause up to 30% of common colds and mild respiratory illnesses [6]. Other CoVs are widespread in both domestic and wild animals, with several posing significant economic impact on livestock and poultry industries.Following the emergence of SARS, two additional human CoVs associated with upper and lower respiratory tract diseases were identified. Three groups independently identified in young children what is likely a single CoV species, and this new CoV has been variously designated NL63,. The second new CoV was discovered in an elderly patient * Corresponding author: Mailing Address: Timothy C. Umland, Ph.D., Hauptman-Woodward Medical Research Institute, 700 Ellicott St., Buffalo, NY 14203-1102, E-mail: umland@hwi.buffalo.edu, Phone: 716-898-8632, Fax: 716-898-8660. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please ...

Human G-CSF synthesis using stress-responsive bacterial proteins

“…Recent studies also verified the proper folding of recombinant hG-CSF using CD spectra (Bae et al, 1998(Bae et al, , 1999Jeong & Lee, 2001). In the other recent studies using CD spectrum, properly folded conformations of recombinant human and viral proteins were structurally analyzed (Leopoldino et al, 2006;Brucz et al, 2007;Tolun et al, 2007;Vamvakas et al, 2007). The CD spectra of the purified fusion-free hG-CSF (Fig.…”