High-level expression, purification and characterization of codon-optimized recombinant hemagglutinin 5 proteins in mammalian cells.
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.


CONCLUSIONS
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.
Yang
Jing-lin
JL
Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Wang
Hong-liang
HL
Wang
Shun-xin
SX
Yang
Peng
P
Liu
Kang-tai
KT
Jiang
Cheng-yu
CY
D002460:Cell Line; D003062:Codon / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
D003062:Codon; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011994:Recombinant Proteins