High-level expression, purification and characterization of codon-optimized recombinant hemagglutinin 5 proteins in
Jing-lin Yang1, Hong-liang Wang, Shun-xin Wang
1Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Background:
Numerous Asian cases of avian influenza virus infection, especially the highly pathogenic strain H5N1, in humans have raised the concern that another influenza pandemic is close. However, there are no effective therapeutic drugs or preventative vaccines available. Hemagglutinin is the membrane glycoprotein of avian influenza virus responsible for receptor binding to human cells and the main immunogenic protein that elicits a strong immune response. Although this protein is of great importance to the study of pathogenesis and vaccine development, its expression and purification are difficult due to high levels of glycosylation.
Methods:
In this study, we expressed codon-optimized, full-length hemagglutinin 5 (H5) protein fused with a human IgG Fc tag (H5-Fc) in HEK293 cells. To enhance secretion of this protein, we also deleted the transmembrane domain and the intracellular domain of the H5 protein (H5DeltaTM-Fc). Purified proteins were obtained using a protein A column.
Results:
ELISA revealed that the yield of soluble H5DeltaTM-Fc protein in the supernatant was about 20 mg/L. Western blotting and fluorescence activated cell sorter (FACS) indicated that the purified H5 protein was correctly folded and biologically active.
Conclusion:
Purification of H5 proteins from mammalian cells could be used for large-scale production of recombinant H5 protein for basic scientific research or the development of vaccines.
Insights
Researchers developed a method to produce large quantities of purified H5 hemagglutinin (H5) protein from avian influenza virus. This advancement aids in developing potential vaccines and understanding H5 pathogenesis.
Area of Science:
- Virology
- Immunology
- Biotechnology
Background:
- Avian influenza virus, particularly H5N1, poses a pandemic threat.
- Effective vaccines and therapeutics are currently unavailable.
- Hemagglutinin (H5) is crucial for viral entry and is a key immunogen, but difficult to purify due to glycosylation.
Purpose of the Study:
- To develop an efficient method for expressing and purifying functional H5 hemagglutinin protein.
- To facilitate large-scale production of H5 protein for research and vaccine development.
Main Methods:
- Codon-optimized, full-length H5 hemagglutinin fused with an Fc tag (H5-Fc) was expressed in HEK293 cells.
- Transmembrane and intracellular domains were deleted to enhance secretion (H5DeltaTM-Fc).
- Protein A affinity chromatography was used for purification.
Main Results:
- Soluble H5DeltaTM-Fc protein yield reached approximately 20 mg/L.
- Western blotting and FACS confirmed correct folding and biological activity of the purified H5 protein.
Conclusions:
- Mammalian cell expression and purification provide a viable strategy for large-scale recombinant H5 protein production.
- This method supports basic scientific research and the development of H5-based vaccines.
