A new insect cell glycoengineering approach provides baculovirus-inducible glycogene expression and increases human-type glycosylation efficiency.
Insect cells are often glycoengineered using DNA constructs encoding foreign glyocoenzymes under the transcriptional control of the baculovirus immediate early promoter, ie1. However, we recently found that the delayed early baculovirus promoter, 39K, provides inducible and higher levels of transgene expression than ie1 after baculovirus infection (Lin and Jarvis, 2013). Thus, the purpose of this study was to assess the utility of the 39K promoter for insect cell glycoengineering. We produced two polyclonal transgenic insect cell populations in parallel using DNA constructs encoding foreign glycoenzymes under either ie1 (Sfie1SWT) or 39K (Sf39KSWT) promoter control. The surface of Sfie1SWT cells was constitutively sialylated, whereas the Sf39KSWT cell surface was only strongly sialylated after baculovirus infection, indicating Sf39KSWT cells were inducibly-glycoengineered. All nine glycogene-related transcript levels were induced by baculovirus infection of Sf39KSWT cells and most reached higher levels in Sf39KSWT than in Sfie1SWT cells at early times after infection. Similarly, galactosyltransferase activity, sialyltransferase activity, and sialic acid levels were induced and reached higher levels in baculovirus-infected Sf39KSWT cells. Finally, two different recombinant glycoproteins produced by baculovirus-infected Sf39KSWT cells had lower proportions of paucimannose-type and higher proportions of sialylated, complex-type N-glycans than those produced by baculovirus-infected Sfie1SWT cells. Thus, the 39K promoter provides baculovirus-inducible expression of foreign glycogenes, higher glycoenzyme activity levels, and higher human-type N-glycan processing efficiencies than the ie1 promoter, indicating that this delayed early baculovirus promoter has great utility for insect cell glycoengineering.
Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA.
Ann M
AM
Toth
Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan.
Chu-Wei
CW
Kuo
Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan.
Kay-Hooi
KH
Khoo
Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA. Electronic address: dljarvis@uwyo.edu.
Donald L
DL
Jarvis
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D049109:Cell Proliferation; D004921:Erythropoietin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D019076:Transgenes
D006023:Glycoproteins; D011994:Recombinant Proteins; D004921:Erythropoietin; D019158:N-Acetylneuraminic Acid
Baculovirus expression vector system; Baculovirus promoters; Glycoengineering; Insect cell glycosylation