Expression and secretion of rice alpha-amylase by Saccharomyces cerevisiae.
We report the high level expression and secretion of rice alpha-amylase isozyme by Saccharomyces cerevisiae. Transcription of this gene was under control of the yeast enolase promoter. The synthesized protein had an approximate molecular size of 45 kDa and a pI of approx 4.7 to 5.0. The rice alpha-amylase signal peptide was recognized and efficiently processed by yeast and the active, glycosylated enzyme was secreted into the culture media. This enzyme was purified to homogeneity by affinity chromatography and its enzymatic properties were characterized. The Km and Vmax were found to be similar to those of alpha-amylases from other organisms. The high level of secretion observed in these studies may be due to the unique features of the rice signal peptide and/or to the glycosylation of the recombinant enzyme.
Kumagai
M H
MH
Biosource Genetics Corporation, Vacaville, CA 95688.
Shah
M
M
Terashima
M
M
Vrkljan
Z
Z
Whitaker
J R
JR
Rodriguez
R L
RL
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004247:DNA / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D000516:alpha-Amylases / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification
D011994:Recombinant Proteins; D004247:DNA; D000516:alpha-Amylases