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W2079289360 abstract "The development of efficient methods for production and purification of plant seed storage proteins in heterologous microbial hosts would facilitate structure-function studies of these proteins. This report describes such methods applied to the production and isolation of wheat α-gliadin, a prolamine-type seed storage protein, from Saccharomyces cerevisiae. Beginning with the vector, growth conditions, and extraction methods of Neill et al. [Gene 55 (1987) 303–317], we implemented several improvements to increase the yields of α-gliadin per volume of yeast cell culture. The CYC1::Gli-A2-Y transcriptional fusion vector, pAY31 (Neill et al., 1987), was modified by replacing the ARS1 region of replication with that of the 2 μ plasmid of yeast. We formulated a new medium, a derivative of synthetic defined (SD) medium supplemented with several nitrogen sources, that allows both selection for maintenance of plasmids and growth to high cell densities. Stationary phase cultures of cells bearing the modified expression vector, and grown in this medium with glycerol and lactate as carbon sources, contain significantly higher levels of α-gliadin than log-phase cultures grown in SD glucose. Sonication in 80% ethanol selectively and efficiently extracts the α-gliadin from cell pellets of small- or large-scale cultures, allowing the purification of several hundred μg of the wheat protein per liter injust a few high-yield steps. The α-gliadin isolated from yeast elutes at the same position in HPLC as the A-gliadin fraction purified from wheat flour. N-terminal amino acid (aa) sequencing reveals that the signal peptide is removed from the gliadin precursor in yeast cells. About two-thirds of the molecules have the same N terminus as A-gliadin made in wheat endosperm cells. The remaining third of the α-gliadin is apparently cleaved at a similar processing site three aa closer to the N terminus of the prepeptide." @default.
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W2079289360 date "1992-07-01" @default.
W2079289360 modified "2023-10-17" @default.
W2079289360 title "Purification and characterization of wheat α-gliadin synthesized in the yeast, Saccharomyces cerevisiae" @default.
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W2079289360 doi "https://doi.org/10.1016/0378-1119(92)90507-l" @default.
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