Saccharomyces cerevisiae와 Pichia pastoris에서 Bovine Pancreatic Deoxyribonuclease I의 과발현과 특성
Overexpression and Characterization of Bovine Pancreatic Deoxyribonuclease I in Saccharomyces cerevisiae and Pichia pastoris
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본 연구에서는 S. cerevisiae와 P. pastoris에서 bovine pancreatic (bp-) DNase I의 과발현과 재조합 DNase I의 특성을 조사하였다. bp-DNase I 유전자는 GAL10 promoter, MFα, GAL7 terminator 사이에 삽입하여 재조합 plasmid인pGAL-MFα-DNaseI (6.4 kb)를 구축하였다. 그리고 bp-DNase I 유전자를 AOX1 promoter, MFα, AOX1 terminator 에 삽입하여 재조합 plasmid인 pPEXI (8.8 kb)를 구축하였다. 재조합 plasmid인 pGAL-MFα-DNaseI과 pPEXI를 각각 S. cerevisiae와 P. pastoris 숙주세포에 형질전환시켰다. 형질전환된 효모세포들을 galactose와 methanol 배지에서30oC, 48시간 배양하면 bp-DNase I은 대부분이 배양 상등액으로 과발현되었다. P. pastoris 형질전환체는 배양 상등액에서 45.5 unit/mL의 DNase I 활성을 보였으며, 반면에 S. cerevisiae 형질전환체는 37.7 unit/mL의 DNase I 활성을 보였다. 또한 DNA 분해 특성을 조사한 결과, P. pastoris 재조합 DNase I으로 기질 DNA(calf thymus)를 처리하였을 때1분 이내 DNA가 분해되는 것을 확인할 수 있었으며 이는상업용 bp-DNase I과 S. cerevisiae 재조합 DNase I으로 처리했을 때보다 빠른 분해 패턴을 보였다.
Abstract
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 (8.8kb). 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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