[Enhancement of Coprinus cinereus peroxidase in Pichia pastoris by co-expression chaperone PDI and Ero1].
The 1,095 bp gene encoding peroxidase from Coprinus cinereus was synthesized and integrated into the genome of Pichia pastoris with a highly inducible alcohol oxidase. The recombinant CiP (rCiP) fused with the a-mating factor per-pro leader sequence derived from Saccharomyces cerevisiae was secreted into the culture medium and identified as the target protein by mass spectrometry, confirming that a C. cinereus peroxidase (CiP) was successfully expressed in P. pastoris. The endoplasmic reticulum oxidoreductase 1 (Ero1) and protein disulfide isomerase (PDI) were co-expressed with rCiP separately and simultaneously. Compared with the wild type, overexpression of PDI and Erol-PDI increaseed Cip activity in 2.43 and 2.6 fold and their activity reached 316 U/mL and 340 U/mL respectively. The strains co-expressed with Erol-PDI was used to high density fermentation, and their activity reached 3,379 U/mL, which was higher than previously reported of 1,200 U/mL.
Fei
F
Chen
Meirong
M
Hu
Xianzhang
X
Jiang
Yong
Y
Tao
Jianzhong
J
Huang
D003302:Coprinus / Q000201:enzymology*; D003470:Culture Media; D003593:Cytoplasm; D005285:Fermentation; D006023:Glycoproteins / Q000378:metabolism*; D013058:Mass Spectrometry; D000072235:Mating Factor; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000378:metabolism*; D010455:Peptides; D010544:Peroxidases / Q000096:biosynthesis*; D010843:Pichia / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D017510:Protein Folding; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
D003470:Culture Media; D006023:Glycoproteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D000072235:Mating Factor; D010544:Peroxidases; D050862:Oxidoreductases Acting on Sulfur Group Donors; C113186:ERO1 protein, S cerevisiae; D019704:Protein Disulfide-Isomerases