Expression of oryzacystatin cDNA using yeast artificial chromosome under ADH promoter in baker's yeast

We constructed an expression vector into a yeast artificial chromosome (YAC) harboring the cDNA for oryzacystatin, a cysteine proteinase inhibitor from rice, under the control of the yeast ADH promoter. When the expression vector was introduced into Saccharomyces cerevisiae in the form of either an artificial chromosome or a circular plasmid, transformants carrying the DNA grew well in a selective medium. However, the content of the introduced DNA decreased significantly during passages in non-selective YPD medium. The stability of the introduced DNA was enhanced in selected clones obtained as colonies viable in selective medium after many passages in YPD medium. The stable transformants thus obtained expressed the mRNA for oryzacystatin at levels as high as those of intrinsic yeast ADH.