Expression, processing and secretion of a proteolytically-sensitive insect diuretic hormone by Saccharomyces cerevisiae requires the use of a yeast strain lacking genes encoding the Yap3 and Mkc7 endoproteases found in the secretory pathway.
A system is described for the heterologous expression of peptides in Saccharomyces 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 treated with peptidylglycine alpha-amidating enzyme to generate the alpha-amidated, mature, form of Mas-DH or Mas-DH[K22Q], which were biologically active. Successful expression of full-length Mas-DH+Gly depended upon the use of a protease-deficient yeast strain. In wild-type strains, Mas-DH+Gly was recovered only as proteolytic fragments, even in the presence of various protease inhibitors. Expression of Mas-DH+Gly in strains deficient in either the Mkc7 or the Yap3 protease reduced proteolysis, while no proteolysis of Mas-DH+Gly was detectable in a strain lacking both proteases. This protease-deficient strain may prove of general utility for expression of peptides. Analysis of recovered proteolytic fragments revealed a complex pattern of cleavage sites. Both the Yap3 and Mkc7 proteases preferred to cleave at a single Glu-Lys downward 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 specificity of Yap3 and Mkc7 expressed at physiological levels.
Copley
K S
KS
Department of Biochemistry, University of Nevada, Reno, NV 89557, USA.
Alm
S M
SM
Schooley
D A
DA
Courchesne
W E
WE
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics; D017353:Gene Deletion; D005800:Genes, Fungal; D017344:Genes, Insect; D005813:Genes, Synthetic; D005973:Glutamine; D007301:Insect Hormones / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D008239:Lysine; D008297:Male; D018402:Manduca / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D005656:Fungal Proteins; D007301:Insect Hormones; D010446:Peptide Fragments; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C109616:diuretic hormone, Manduca sexta; D005973:Glutamine; D000242:Cyclic AMP; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases; C499266:YPS1 protein, S cerevisiae; D008239:Lysine