Optimization of the functional expression of Coprinus cinereus peroxidase in Pichia pastoris by varying the host and promoter.
Peroxidase from Coprinus cinereus (CiP) has attracted attention for its high specific activity and broad substrate spectrum compared with other peroxidases. In this study, the functional expression of this peroxidase was successfully achieved in the methylotrophic yeast Pichia pastoris. The expression level of CiP was increased by varying the microbial hosts and the expression promoters. Since a signal sequence, such as the alpha mating factor of Saccharomyces cerevisiae, was placed preceding the cDNA of the CiP coding gene, expressed recombinant CiP (rCiP) was secreted into the culture broth. The Mut+ Pichia pastoris host showed a 3-fold higher peroxidase activity, as well as 2-fold higher growth rate, compared with the Muts Pichia pastoris host. Furthermore, the AOX1 promoter facilitated a 5-fold higher expression of rCiP than did the GAP promoter.
Kim
Su-Jin
SJ
Korea Research Institute of Chemical Technology, 100 Jang-dong, Yuseong-gu, Daejeon 305-343, Korea.
Lee
Jeong-Ah
JA
Kim
Yong-Hwan
YH
Song
Bong-Keun
BK
D002455:Cell Division; D003302:Coprinus / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D007700:Kinetics; D010544:Peroxidases / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism
D018076:DNA, Complementary; D004271:DNA, Fungal; D011994:Recombinant Proteins; D010544:Peroxidases