Baculovirus expression of alkaline phosphatase as a reporter gene for evaluation of production, glycosylation and secretion.
We have devised a simple and efficient baculovirus expression vector system to evaluate insect tissue culture cells for their capacity to express, glycosylate and secrete foreign proteins. A truncated placental alkaline phosphatase (SEAP) gene was inserted into the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome under the transcriptional control of the polyhedrin gene promoter. Production levels, glycosylation, and secretion of the recombinant protein were examined in Trichoplusia ni (BTI-TN-5B1-4) and Spodoptera frugiperda (Sf9) cell lines. The assay for SEAP activity, which is fast, inexpensive, and quantitative to concentrations of 20 picograms per milliliter, was used to assess cell-associated and secreted SEAP activity. The proportion of SEAP which is modified with N-linked oligosaccharide can also be determined due to the difference in mobilities during SDS-PAGE between the glycosylated and nonglycosylated forms of the protein.
Boyce Thompson Institute for Plant Research, Ithaca, New York 14853.
T R
TR
Davis
K M
KM
Trotter
R R
RR
Granados
H A
HA
Wood
D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000737:chemistry; D000818:Animals; D016367:Baculoviridae; D002478:Cells, Cultured / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006031:Glycosylation; D007313:Insecta / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D011994:Recombinant Proteins; D000469:Alkaline Phosphatase