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KMID : 1234420120400040348
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Korean Journal of Microbiololgy and Biotechnology
2012 Volume.40 No. 4 p.348 ~ p.355
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Overexpression and Characterization of Bovine Pancreatic Deoxyribonuclease I in Saccharomyces cerevisiae and Pichia pastoris
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Cho Eun-Soo
Kim Jeong-Hwan
Yoon Ki-Hong
Kim Yeon-Hee
Nam Soo-Wan
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Abstract
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In the present study, we investigated the overexpression and characterization of bovine pancreatic (bp)- DNase I in Saccharomyces cerevisiae and Pichia pastoris. The bp-DNase I gene was fused in frame with the GAL10 promoter, MFα, and GAL7 terminator sequences, resulting in the plasmid, pGAL-MFα-DNaseI (6.4 kb). Also, the bp-DNase I gene was fused in frame with the AOX1 promoter, MFα, and AOX1 terminator sequences, resulting in the plasmid, pPEXI (8.8 kb). The recombinant plasmids, pGAL-MFα-DNaseI and pPEXI were introduced into S. cerevisiae and P. pastoris host cells, respectively. When the transformed yeast cells were cultured at 30oC for 48 h in galactose or methanol medium, bp-DNase I was overexpressed and the most of activity was found in the extracellular fraction. P. pastoris transformant activity showed 45.5 unit/mL in the culture medium at 48 h cultivation, whereas S. cerevisiae transformant revealed 37.7 unit/mL in the extracellular fraction at 48 h cultivation. The enzymatic characteristics, such as DNA cleavage and half life were investigated. Treatment of the recombinant DNase I from P. pastoris induced degradation of the calf thymus DNA within 1 minute, and this DNA degradation rate was higher than that of commercial bp-DNase I (SIGMA) and the recombinant DNase I from S. cerevisiae.
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KEYWORD
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Bovine Pancreatic Deoxyribonuclease I, DNA degradation, Saccharomyces cerevisiae, Pichia pastoris, MFα signal
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