ABSTRACT
Saccharomyces cerevisiae is attractive as a producer of heterologous proteins. Heterologous protein secretion is often more efficient when secretion signals from yeast are used, such as those of a factor precursor, invertase and acid phosphatase. Chloramphenicol inhibits protein synthesis carried out by 70S ribosomes of prokaryotes and mitochondria, but not by 80S ribosomes found in the cytoplasm of eukaryotic cells such as yeast. For high-level expression of heterologous proteins, replicating vectors appear attractive because some of them exist at high copy numbers. A chloramphenicol acetyltransferase (CAT) expression cassette has been constructed by inserting the CAT coding sequence, and its associated bacterial ribosome-binding site, between a modified alcohol dehydrogenase promoter and a iso-1-cytochrome c terminator. The expression cassette makes use of the glyceraldehyde-3-phosphate dehydrogenase promoter and the ARG3 terminator. When high levels of a heterologous protein are toxic for the yeast cell, the use of a regulated promoter is of crucial importance.
