Purification of respiratory syncytial virus F and G proteins.
Respiratory syncytial virus (RSV) is the most important cause of severe lower respiratory tract infections of infants in industrial nations. In addition, the participation of RSV in the genesis of asthma is under discussion. The RSV glycoproteins F and G have key positions in the viral pathogenesis. At present no satisfactory protein purification protocols are available for these proteins. The methods published for the G protein using preparative SDS-PAGE or immunoaffinity chromatography yield only small amounts of purified G protein that has partially lost its antigenicity. We describe a three-step purification protocol for these glycoproteins. RSV-infected HEp-2 cells were lysed by a Triton X-100 containing buffer. The viral proteins were captured by QAE-Sephadex A-50 material in a batch procedure. A first elution with 100 mM NaCl led to a crude F protein fraction, and a second elution with 300 mM NaCl led to a crude G protein fraction. The F protein was further purified on a Lentil-lectin Sepharose 4B column and finally polished using a Resource Isopropyl column. Lentil-lectin Sepharose 4B was also used to purify the G protein from the crude fraction, but polishing of the G protein was carried out on a Resource Q column. Homogenous RSV-F and RSV-G proteins were obtained by this protein purification protocol. No loss of antigenicity could be observed during this procedure as the highly purified viral proteins remain detectable by a set of monoclonal antibodies and specific antisera. The G protein was isolated as a 90000 monomer, whereas the purified F protein was recovered as a functional homodimer of 140000.
Roder
C
C
Department of Medical Microbiology and Virology, Ruhr University, Bochum, Germany. christian.roder@ruhr-uni-bochum.de
Krusat
T
T
Reimers
K
K
Werchau
H
H
D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D015707:HN Protein; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000302:isolation & purification*
D015707:HN Protein; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C116204:attachment protein G