EXPRESSION, PROCESSING AND SECRETION OF A PROTEOLYTICALLY-SENSITIVE INSECT DIURETIC HORMONE BY SACCHAROMYCES-CEREVISIAE REQUIRES THE USE OFA YEAST-STRAIN LACKING GENES ENCODING THE YAP3 AND MKC7 ENDOPROTEASESFOUND IN THE SECRETORY PATHWAY

A system is described for the heterologous expression of peptides in S accharomyces cerevisiae. A synthetic gene encoding a precursor of the 41 amino acid Manduca sexta diuretic hormone (Mas-DH) was expressed at 0.8 mg/l purified peptide. A precursor of a mutant peptide of Mas-DH, Mas-DH[K22Q] was also expressed. The peptides were purified, then tre ated with peptidylglycine alpha-amidating enzyme to generate the alpha -amidated, mature, form of Mas-DH or Mas-DH[K22Q], which were biologic ally active. Successful expression of full-length Mas-DH + Gly depende d upon the use of a protease-deficient yeast strain. In wild-type stra ins, Mas-DH + Gly was recovered only as proteolytic fragments, even in the presence of various protease inhibitors. Expression of Mas-DH + G ly in strains deficient in either the Mkc7 or the Yap3 protease reduce d proteolysis, while no proteolysis of Mas-DH+Gly was detectable in a strain lacking both proteases. This protease-deficient strain may prov e of general utility for expression of peptides. Analysis of recovered proteolytic fragments revealed a complex pattern of cleavage sites. B oth the Yap3 and Mkc7 proteases preferred to cleave at a single Glu-Ly s down arrow-Glu-Arg site. Analysis of secondary cleavage sites showed that Yap3 preferred to cleave after either Lys or Arg and Mkc7 after Lys. This paper is the first report on the in vivo activity and specif icity of Yap3 and Mkc7 expressed at physiological levels.