title: Effects of signal sequences and folding accessory proteins on extracellular expression of carboxypeptidase Y in recombinant Saccharomyces cerevisiae.
abstract: Carboxypeptidase Y (CPY) is a yeast vacuolar protease with useful applications including C-terminal sequencing of peptides and terminal modification of target proteins. To overexpress CPY with the pro-sequence (proCPY) encoded by the Saccharomyces cerevisiae PRC1 gene in recombinant S. cerevisiae, the proCPY gene was combined with the gene coding for a signal sequence of S. cerevisiae mating factor α (MFα), invertase (SUC2), or Kluyveromyces marxianus inulinase (INU1). Among the three constructs, the MFα signal sequence gave the best specific activity of extracellular CPY. To enhance the CPY expression level, folding accessory proteins of Kar2p, Pdi1p and Ero1p located in the S. cerevisiae endoplasmic reticulum were expressed individually and combinatorially. A single expression of Kar2p led to a 28 % enhancement in extracellular CPY activity, relative to the control strain of S. cerevisiae CEN.PK2-1D/p426Gal1-MFαCPY. Coexpression of Kar2p, Pdi1p and Ero1p gave a synergistic effect on CPY expression, of which activity was 1.7 times higher than that of the control strain. This work showed that engineering of signal sequences and protein-folding proteins would be helpful to overexpress yeast proteins of interest.
authors.affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul, 151-921, Korea.
authors.forename: So-Yeon
authors.initials: SY
authors.lastname: Shin
authors.forename: Yi-Hyun
authors.initials: YH
authors.lastname: Bae
authors.forename: Sun-Ki
authors.initials: SK
authors.lastname: Kim
authors.forename: Yeong-Je
authors.initials: YJ
authors.lastname: Seong
authors.forename: Suk-Hwan
authors.initials: SH
authors.lastname: Choi
authors.forename: Kyoung Heon
authors.initials: KH
authors.lastname: Kim
authors.forename: Yong-Cheol
authors.initials: YC
authors.lastname: Park
authors.forename: Jin-Ho
authors.initials: JH
authors.lastname: Seo
mesh_terms: D043402:Cathepsin A / Q000096:biosynthesis* / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000235:genetics / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011498:Protein Precursors / Q000378:metabolism; D021382:Protein Sorting Signals / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D006023:Glycoproteins; D018840:HSP70 Heat-Shock Proteins; C060153:KAR2 protein, yeast; C048632:MF(ALPHA)1 protein, S cerevisiae; C080576:PDI1 protein, S cerevisiae; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C113186:ERO1 protein, S cerevisiae; D043402:Cathepsin A; C499283:PRC1 protein, S cerevisiae; D019704:Protein Disulfide-Isomerases