Purification and characterization of wheat alpha-gliadin synthesized in the yeast, Saccharomyces cerevisiae.
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 alpha-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 alpha-gliadin per volume of yeast cell culture. The CYCl::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 mu 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 alpha-gliadin than log-phase cultures grown in SD glucose. Sonication in 80% ethanol selectively and efficiently extracts the alpha-gliadin from cell pellets of small- or large-scale cultures, allowing the purification of several hundred micrograms of the wheat protein per liter in just a few high-yield steps. The alpha-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Blechl
A E
AE
U.S. Department of Agriculture, Western Regional Research Center, Albany, CA 94710.
Thrasher
K S
KS
Vensel
W H
WH
Greene
F C
FC
D000595:Amino Acid Sequence; D003470:Culture Media; D005786:Gene Expression Regulation; D005903:Gliadin / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009584:Nitrogen; D021382:Protein Sorting Signals / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D013010:Sonication; D014908:Triticum
D003470:Culture Media; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D005903:Gliadin; D009584:Nitrogen