Journal of the Korean Wood Science and Technology

The Korean Society of Wood Science & Technology (한국목재공학회)
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Expression of a Manganese Peroxidase Gene (mnp5) from White rot fungus Phanerochaete chrysosporium in the Pichia pastoris

백색부후균 Phanerochaete chrysosporium에서 유래한 Manganese Peroxidase Gene(mnp5)의 Pichia pastoris에서의 이종발현

  • Lee, Jae-Won (Department of Forest Sciences, College of Agriculture & Life Sciences, Seoul National University) ;
  • Yang, In (Department of Forest Sciences, College of Agriculture & Life Sciences, Seoul National University) ;
  • Igarashi, Kiyohiko (Graduate School of Agriculture and Life Sciences, The University of Tokyo) ;
  • Samejima, Masahiro (Graduate School of Agriculture and Life Sciences, The University of Tokyo) ;
  • Choi, In-Gyu (Department of Forest Sciences, College of Agriculture & Life Sciences, Seoul National University)
  • 이재원 (서울대학교 농업생명과학대학 산림과학부) ;
  • 양인 (서울대학교 농업생명과학대학 산림과학부) ;
  • 五十嵐圭日子 (동경대학교 농업생명과학대학원) ;
  • 鮫島正浩 (동경대학교 농업생명과학대학원) ;
  • 최인규 (서울대학교 농업생명과학대학 산림과학부)
  • Received : 2005.05.16
  • Accepted : 2005.06.08
  • Published : 2005.07.25
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Abstract

The manganese peroxidase (mnp5) from white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. The majority of the rMnP5 (recombinant MnP5) produced by P. pastoris exhibited an approximate molecular mass 45 kDa considerably larger than that of the predicting mnp5 due to two glycosylation sites of mnp5. After site direct mutation treatment, the effect of N-linked hyperglycosylation was examined by enzyme activity. Analysis by sodium dodesyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with a molecular mass of 37 kDa. Enzyme activity of M-rMnP5 (mutant recombinant MnP5) was similar to that of rMnP5, indicating that hyperglycosylation did not affect the active site. In this work, active mnp5 was successfully expressed in P. pastoris, suggesting that P. pastoris has potential capability of producing active heme-containing proteins.

백색부후균 Phanerochaete chrysosporium으로부터 유래한 Manganese peroxidase (mnp5)를 methylotrophic yeast인 Pichia pastoris에서 이종 발현을 하였다. 이종발현으로부터 얻어진 단백질은 클로닝으로부터 예상되어지는 분자량보다 높은 분자량인 45 kDa으로 나타났다. 이것은 mnp5가 가지고 있는 glycosylation site에 의한 것이며, N-linked hyperglycosylation이 효소 활성에 영향을 미치는지를 site direct mutation에 의해 확인하였다. Sodium dodesyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)와 Coomassie Brilliant Blue (CBB) 염색에 의해 분자량을 확인한 결과 약 37 kDa으로 나타났으며, 효소활성을 측정한 결과 glycosylation이 효소 활성에 영향을 미치지 않는 것으로 나타났다. 따라서 본 연구로부터 P. pastoris에서 mnp5의 이종발현이 성공적으로 이루어졌으며 이러한 결과로부터 heme을 포함하고 있는 단백질의 이종발현 생산의 가능성을 보여주었다.

Keywords

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