Expression,glycosylation and secretion of phaseolin in a baculovirus system |
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Authors: | Mauricio M Bustos Verne A Luckow Lawrence R Griffing Max D Summers Timothy C Hall |
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Institution: | (1) Department of Biology, Texas A&M University, 77843-3258 College Station, TX, USA;(2) Department of Entomology, Texas A&M University, 77843-3258 College Station, TX, USA |
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Abstract: | In this report, we describe the efficient expression and glycosylation, in insect cells, of -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/106 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. |
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Keywords: | baculovirus eukaryotic expression vectors phaseolin plant glycoproteins protein targeting |
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