title: Expression and purification of a chimeric protein consisting of the ectodomains of M and GP5 proteins of porcine reproductive and respiratory syndrome virus (PRRSV).
abstract: Porcine reproductive and respiratory syndrome (PRRS) is the most economically important infectious disease currently affecting the swine industry worldwide. In the US alone, it causes economic losses of more than 560 million dollars every year. Although killed-virus and modified-live PRRS vaccines are commercially available, the unsatisfactory efficacy and safety of current vaccines drives the impetus of developing novel PRRSV vaccines. To fulfill this purpose, we designed a chimeric protein consisting of the ectodomains of viral GP5 and M protein, the two most widely studied subunit vaccine targets, and expressed it in E. coli. An optimized purification/refolding process composed of immobilized metal ion affinity chromatography, dialysis refolding and anion exchange chromatography was developed to purify the chimeric protein from the inclusion bodies. This process could recover approximately 12mgprotein/l E. coli broth with near 100% purity and very low endotoxin level. In addition, the purified protein is antigenic, can bind to a cellular receptor for the virus (heparan sulfate), and can block virus infection of susceptible cells. Therefore, the chimeric protein is a promising subunit vaccine candidate against PRRSV.
authors.affiliation: Department of Biological Systems Engineering, Virginia Tech, 200 Seitz Hall, Blacksburg, VA 24061, USA.
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Hu
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Ni
authors.forename: X J
authors.initials: XJ
authors.lastname: Meng
authors.forename: Chenming
authors.initials: C
authors.lastname: Zhang
mesh_terms: D015153:Blotting, Western; D002852:Chromatography, Ion Exchange / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D014759:Viral Envelope Proteins / Q000302:isolation & purification* / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D006497:Heparan Sulfate; D006639:Histidine; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins; C471213:His-His-His-His-His-His; C525999:M protein, porcine reproductive and respiratory syndrome virus; C114789:glycoprotein 5, PRRSV