Purification of the major envelop protein GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) from native virions.
Porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of an economically important swine disease that has been devastating the global swine industry since the early 1990s. The current PRRSV vaccines are not very effective largely due to heterogeneic nature of the virus. The major envelope protein, GP5, exposes outside the virion, induces neutralizing antibodies, and thus is a primary target for developing a subunit vaccine. In this study, we report a process for purification of GP5 protein from native virions of PRRSV propagated in MARC-145 cells. PRRSV virions were first purified and concentrated through sucrose cushion ultracentrifugation. GP5 protein was subsequently solubilized with Triton X-100 detergent for further processing. Cation exchange chromatography (CEX) was utilized for partial fractionation of GP5, although the viral nucleocapsid protein (N) was a major impurity in CEX elution fractions. During a second chromatographic step, hydrophobic interaction chromatography (HIC) further purified GP5 protein by means of a two-stage elution scheme. Pure GP5 protein was eluted from the HIC resin in the second HIC elution stage by Triton X-100 displacement; however the protein is present as a homodimeric/tetrameric aggregate. This process may be useful in PRRSV subunit vaccine development.
Matanin
Brad M
BM
Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 200 Seitz Hall, Blacksburg, VA 24061, USA.
Huang
Yaowei
Y
Meng
X J
XJ
Zhang
Chenming
C
D000818:Animals; D002460:Cell Line; D002845:Chromatography / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods; D057927:Hydrophobic and Hydrophilic Interactions; D019316:Porcine respiratory and reproductive syndrome virus / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology; D014759:Viral Envelope Proteins / Q000276:immunology / Q000302:isolation & purification*; D014765:Viral Vaccines; D014771:Virion / Q000737:chemistry; D014776:Virus Cultivation
D014759:Viral Envelope Proteins; D014765:Viral Vaccines