title: Expression, glycosylation and secretion of phaseolin in a baculovirus system.
abstract: In this report, we describe the efficient expression and glycosylation, in insect cells, of β-phaseolin polypeptides (M r 45 and 48 kDa) from Phaseolus vulgaris, by means of a baculovirus expression vector. N-terminal sequence analysis demonstrated that the signal peptide was efficiently processed. Tunicamycin treatment suppressed both phaseolin bands seen in untreated or control cells, and resulted in a single species (M r 43 kDa). We provide evidence that the observed size heterogeneity arises by asymmetric glycosylation of a single, high-molecular weight precursor. These results also indicate that differential glycosylation of phaseolin polypeptides can occur on the product of a single gene, and, in that sense, is not dependent on amino acid sequence variations. Phaseolin accumulates to a very high level (90 µg/10(6) cells), 90% of it being secreted into the culture medium. Immuno-gold staining and electron microscopy demonstrated phaseolin polypeptides in electron-dense, membrane-bound vesicles seen at the periphery of the cytoplasm of infect cells and in cytoplasmic multivesicular bodies. The effect on protein accumulation of a single-basepair transversion (G»C) at position +6 is also described. This study constitutes, to our knowledge, one of the first instances of a plant protein being expressed in insect cells and suggests possible differences in the sorting mechanisms of glycoproteins from legume seeds and those from Spodoptera frugiperda cell line Sf9.
authors.affiliation: Department of Biology, Texas A&M University, 77843-3258, College Station, TX, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Bustos
authors.forename: V A
authors.initials: VA
authors.lastname: Luckow
authors.forename: L R
authors.initials: LR
authors.lastname: Griffing
authors.forename: M D
authors.initials: MD
authors.lastname: Summers
authors.forename: T C
authors.initials: TC
authors.lastname: Hall