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Purification of respiratory syncytial virus F and G proteins
1Department of Medical Microbiology and Virology, Ruhr University, Bochum, Germany. christian.roder@ruhr-uni-bochum.de
Insights
This study presents a novel three-step protocol for purifying Respiratory Syncytial Virus (RSV) glycoproteins F and G. The method yields homogenous proteins without loss of antigenicity, crucial for developing effective RSV vaccines and diagnostics.
Area of Science:
- Virology
- Protein Biochemistry
- Immunology
Background:
- Respiratory Syncytial Virus (RSV) is a major cause of infant respiratory infections.
- RSV glycoproteins F and G are critical for viral pathogenesis.
- Existing purification methods for RSV glycoproteins are inadequate, yielding small amounts of protein with reduced antigenicity.
Purpose of the Study:
- To develop a robust and efficient three-step protocol for purifying RSV F and G glycoproteins.
- To ensure the purified glycoproteins retain their antigenicity for further research and development.
Main Methods:
- RSV-infected HEp-2 cells were lysed and viral proteins captured using QAE-Sephadex A-50.
- Proteins were fractionated by sequential elution with NaCl.
- Further purification utilized Lentil-lectin Sepharose 4B, Resource Isopropyl (for F), and Resource Q (for G) columns.
Main Results:
- A three-step protocol successfully purified homogenous RSV F and G glycoproteins.
- Purified proteins showed no loss of antigenicity, confirmed by antibody and antiserum detection.
- RSV G protein was isolated as a 90,000 monomer; RSV F protein as a 140,000 functional homodimer.
Conclusions:
- The developed protocol provides a reliable method for obtaining highly purified, antigenically intact RSV F and G glycoproteins.
- This advancement is vital for the development of novel diagnostics and effective vaccines against RSV infections.
Abstract:
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.