Secretion of mature mouse interleukin-2 by Saccharomyces cerevisiae: use of a general secretion vector containing promoter and leader sequences of the mating pheromone alpha-factor.
We have constructed a general expression vector which allows the synthesis and secretion of processed gene products in Saccharomyces cerevisiae. This vector contains yeast DNA, including the promoter of the mating pheromone (alpha-factor), its downstream leader sequence, and the TRP5 terminator. A cDNA [encoding mature mouse interleukin-2 (IL-2); Yokota et al., Proc. Natl. Acad. Sci. USA 82 (1984) 68-72] was fused immediately downstream to the alpha-factor leader sequence. The resulting recombinant plasmid directed the synthesis of mature mouse IL-2 in S. cerevisiae, with most of the T-cell growth-factor (TCGF) activity secreted into the culture fluid and extracellular space. TCGF activities in the cell extract, as well as in the culture fluid, increased in parallel with cell growth. Production of mature mouse IL-2 was inhibited by tunicamycin (TM), with precursor molecules accumulating in the cell extract. The precursor was processed accurately at the junction between the alpha-factor peptide leader sequence and the coding sequence downstream, yielding mature IL-2. The Mr of the secreted mouse IL-2 determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was 17 kDal, a value expected for the mature mouse IL-2 polypeptide based on the nucleotide (nt) sequence.
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics*; D005822:Genetic Vectors; D007376:Interleukin-2 / Q000235:genetics* / Q000378:metabolism; D000072235:Mating Factor; D051379:Mice; D010455:Peptides / Q000235:genetics; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012636:Secretory Rate / Q000187:drug effects; D014415:Tunicamycin / Q000494:pharmacology
D005656:Fungal Proteins; D007376:Interleukin-2; D010455:Peptides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D014415:Tunicamycin; D000072235:Mating Factor