| Products/Services Used | Details | Operation |
|---|---|---|
| Peptide Synthesis | Anti-His Affinity Resin (GenScript) was used for purification of filovirus GP1,2 to avoid bovine serotransferase protein contamination. All but two plasmids are based on reference GP sequences: Lloviu virus/M.schreibersii-wt/ ESP/2003/Asturias-Bat86 (“LLOV”; RefSeq #NC_016144), Bundibugyo virus/H.sapiens-tc/UGA/2007/ Butalya-811250 (“BDBV”; RefSeq #NC_014373), Ebola virus/H.sapiens-tc/COD/1976/Yambuku-Mayinga (“EBOV/Yambuku”; RefSeq #NC_002549), EBOV/H.sapiens-wt/GIN/2014/Makona-C07 (“EBOV/Makona”, GenBank #KJ660347), Sudan virus/H.sapiens-tc/UGA/2000/Gulu-808892 (“SUDV”; RefSeq #NC_006432), Taï Forest virus/H.sapiens-tc/CIV/1994/Pauléoula-CI (“TAFV”; RefSeq #NC_014372), Marburg virus/H. sapiens-tc/KEN/1980/Mt. Elgon-Musoke (“MARV”; RefSeq #NC_001608), and Marburg virus/H.sapiens-tc/ AGO/2005/Angola-20051379 (“MARV/Angola”, GenBank #KU978782). All genes were synthesized by Genscript (Piscataway, NJ) or cloned at Biosafety level 2 by the authors from inactivated filovirus cultures. All experiments were performed in Biosafety level 2 conditions.Anti-His Affinity Resin (GenScript) was used for purification of GP1,2s following the manufacturer’s instruction. Briefly, resin was incubated with supernatant and rotated for 30 min and resins were placed into Econo-Pac Disposable Chromatography columns (BioRad, Hercules, CA) and washed with Tris-buffered saline (pH 7.4). Resins were then incubated with hexa-His peptide (GenScript) at a concentration of 0.5 µg/ml. Eluents containing proteins of interest were concentrated using Pierce Protein Concentrator (Thermo Fisher Scientific, Waltham, MA) for downstream use. Antibody dilutions were as follows: BDBV (rabbit polyclonal, IBT Bioservices, Rockville, MD, USA): 1:5,000; EBOV (6D8, courtesy of John Dye, US Army Medical Research Institute of Infectious Diseases, Frederick, MD): 1:10,000; TAFV (rabbit polyclonal, Alpha Diagnostic International, San Antonio, TX): 1:1,000; SUDV (clone 6D11, BEI Resources, Manassas, VA): 1:5,000; MARV (rabbit polyclonal, IBT Bioservices, Rockville, MD): 1:3,000; and THETM His Tag Antibody (mouse monoclonal, Genscript). | Get A Quote |
| Mammalian Expression System | Get A Quote |
Filoviruses are highly virulent pathogens capable of causing severe disease. The glycoproteins of filoviruses are the only virally expressed proteins on the virion surface and are required for receptor binding. As such， they are the main candidate vaccine antigen. Despite their virulence， most filoviruses are not comprehensively characterized， and relatively few commercially produced reagents are available for their study. Here， we describe two methods for production and purification of filovirus glycoproteins in insect and mammalian cell lines. Considerations of expression vector choice， modifications to sequence， troubleshooting of purification method， and glycosylation differences are all impor... More
