Increased expression and secretion of recombinant alpha-amylase in Saccharomyces cerevisiae by using glycerol as the carbon source.
Saccharomyces cerevisiae transformed with plasmids containing the barley alpha-amylase gene was cultured, and enzyme activity and cell density were monitored at various time intervals. Proteins in yeast extract and culture medium were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blots of intra- and extracellular proteins were sequentially probed with anti-amylase antibody and anti-rabbit horseradish peroxidase conjugate, followed by chemiluminescent detection. The enzyme activity of recombinant barley alpha-amylase secreted by the yeast clone DY150[pYEX-Amyl] showed a significant increase when the culture medium included glycerol as the carbon source. The enhancement reached a 4.5-fold increase at 120 hr, and the effect was strain-nonspecific. Intra- and extracellular proteins increased significantly with time in both the yeast clone and the control grown in YEPG (2% yeast extract, 1% bacto-peptone, 2% glycerol). Proteins in YEPD (2% yeast extract,1% bacto-peptone, 2% glucose) and YEPG cultures showed very different band patterns, indicating that the metabolic pathway was altered. Western blot analysis indicated that the recombinant amylase accumulated inside yeast cells, at a relatively low level, compared with that in the culture medium. The transcript level of the alpha-amylase gene was significantly increased in the clone cultured in YEPG. This investigation demonstrates that the use of glycerol as a carbon source for S. cerevisiae enhances the synthesis and secretion of the recombinant enzyme while suppressing cell growth.
Wong
Dominic W S
DW
Western Regional Research Center, USDA-ARS, 800 Buchanan Street, Albany, California 94710, USA. dwsw@pw.usda.gov
Batt
Sarah B
SB
Lee
Charles C
CC
Robertson
George H
GH
D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005990:Glycerol / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D000516:alpha-Amylases; D005990:Glycerol