Expression and characterization of secreted forms of rubella virus E2 glycoprotein in insect cells.
Two different forms of rubella virus E2 glycoproteins were expressed in insect cells: intact wild-type E2 and a soluble form of E2 (E2 delta Tm) glycoprotein, in which the C-terminal membrane-anchor domain was deleted. E2 delta Tm behaved as a secretory protein and was secreted abundantly (5 mg/liter) from insect cells. In contrast to wild-type E2 (36 kDa), E2 delta Tm was secreted into the media and was detected as two species (33 and 30 kDa). Lectin binding assays in conjunction with glycosidase analyses revealed that both intracellular wild-type E2 and E2 delta Tm contained only N-linked glycans, while the two secreted forms of E2 delta Tm were found to differ in their glycosylation, with the 30-kDa form having only N-linked glycans while the 33-kDa species had both N-linked and O-linked glycans. The secreted E2 delta Tm species were purified by precipitation between 20 and 40% saturation with (NH4)2SO4 and retained full antigenicity. The levels of antibodies elicited in mice immunized with purified E2 delta Tm showed that the immunogenicity of secreted E2 delta Tm compared favorably to that of natural virion E2.
Department of Pathology, University of British Columbia, Vancouver, Canada.
N O
NO
Seto
D
D
Ou
S
S
Gillam
D000818:Animals; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D051379:Mice; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics; D012412:Rubella virus / Q000378:metabolism*; D018411:Spodoptera; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism
D006023:Glycoproteins; D014759:Viral Envelope Proteins; C061827:E2 envelope protein, Rubella virus