Journal
BIOPROCESS AND BIOSYSTEMS ENGINEERING
Volume 37, Issue 6, Pages 1065-1071Publisher
SPRINGER
DOI: 10.1007/s00449-013-1078-0
Keywords
Carboxypeptidase Y; Saccharomyces cerevisiae; Signal sequence; Folding accessory protein; Fermentation
Funding
- Ministry for Oceans and Fisheries [12128295500]
- National Research Foundation of Korea (NRF) [NRF-2013R1A2A2A01004806]
- Advanced Biomass R&D Center (ABC) - Korean Ministry of Science, ICT and Future Planning [2010-0029799]
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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 alpha (MF alpha), invertase (SUC2), or Kluyveromyces marxianus inulinase (INU1). Among the three constructs, the MF alpha 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-MF alpha CPY. 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.
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