Secretory pathway limits the enhanced expression of classical swine fever virus E2 glycoprotein in insect cells.
The 3' untranslated region (UTR) is an important element that determines the level of recombinant protein expression via baculovirus vectors. Previous work using chloramphenicol acetyl transferase as reporter has shown that p10-promoter based baculovirus vectors with the authentic p10 3' UTR resulted in higher expression levels than vectors carrying an SV40 early terminator, as part of a lacZ selection cassette. To examine whether a similar increase in expression levels could be obtained for baculovirus-expressed glycoproteins, the classical swine fever virus E2 antigen was used as a model. With the authentic p10 3' UTR higher levels of E2 transcript were found than in the presence of the SV40 terminator. This higher number of transcripts was accompanied by elevated levels of intracellular, non-glycosylated E2 protein. However, the levels of intracellular glycosylated forms of E2 and of extracellular E2 were similar for both type of terminators. These results show that translation of the recombinant mRNA is not the rate limiting step in the expression of glycoproteins, but the downstream processing and secretion of the translation products.
van Oers
M M
MM
Laboratory of Virology, Wageningen University and Research Centre, Binnenhaven 11, 6709, PD Wageningen, The Netherlands. monique.vanoers@viro.dpw.wau.nl
Thomas
A A
AA
Moormann
R J
RJ
Vlak
J M
JM
D020413:3' Untranslated Regions; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D015151:Immunoblotting; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis; D013539:Simian virus 40 / Q000235:genetics; D018411:Spodoptera / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism*
D020413:3' Untranslated Regions; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C087086:glycoprotein E2, classical swine fever virus