Secretion of the respiratory syncytial virus fusion protein from insect cells using the baculovirus expression system.
Sequences derived from the respiratory syncytial virus (RSV) fusion (F) protein were expressed in insect cells as recombinant glutathione-S-transferase (GST)-tagged proteins. The sequence covering the F2 subunit (GST-F2), and a truncated form of the F protein in which the transmembrane domain was removed (GST-F2/F1), were cloned into the baculovirus pAcSecG2T secretory vector. These virus sequences also had the endogenous virus signal sequence removed and replaced with a signal sequence derived from the baculovirus gp67 glycoprotein, which was present in pAcSecG2T. The recombinant RSV glycoproteins were successfully detected in expressing cells by immunofluorescence assay and in the tissue culture medium by western blot analysis. The secreted recombinant GST-F2/F1 protein was further analysed using glycosidases. Our results showed that the GST-F2/F1 protein were sensitive to peptide:N-glycosidase F (PNGase F) treatment, but not to Endoglycosidase H (EndoH) treatment. This indicates that the secreted recombinant proteins were modified by the addition of mature N-linked glycan chains.
Tan
Boon-Huan
BH
Virology Group Defence Medical and Environmental Research Institute, DSO National Laboratories, Singapore.
Brown
Gaie
G
Sugrue
Richard J
RJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012136:Respiratory Syncytial Viruses / Q000235:genetics; D018411:Spodoptera; D014760:Viral Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D014760:Viral Fusion Proteins