Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cell Surface Display of Arylsulfatase Gene from Pseudoalteromonas carageenovora in Saccharomyces cerevisiae

Saccharomyces cerevisiae에서 Pseudoalteromonas carageenovora 유래 Arylsulfatase 유전자의 표층 발현

  • Cho, Eun-Soo (Department of Biotechnology and Bioengineering, Dong-Eui University) ;
  • Kim, Hyun-Jin (Department of Biomaterial Control, Dong-Eui University) ;
  • Jung, So-A (Department of Biotechnology and Bioengineering, Dong-Eui University) ;
  • Kim, Jeong-Hwan (Department of Biomaterial Control, Dong-Eui University) ;
  • Kim, Yeon-Hee (Department of Biotechnology and Bioengineering, Dong-Eui University) ;
  • Nam, Soo-Wan (Department of Biotechnology and Bioengineering, Dong-Eui University)
  • 조은수 (동의대학교 생명공학과) ;
  • 김현진 (동의대학교 바이오물질제어학과) ;
  • 정소아 (동의대학교 생명공학과) ;
  • 김정환 (동의대학교 바이오물질제어학과) ;
  • 김연희 (동의대학교 생명공학과) ;
  • 남수완 (동의대학교 생명공학과)
  • Published : 2009.12.28
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Abstract

In this study, the arylsulfatase gene (astA, 984 bp ORF) from Pseudoalteromonas carrageenovora genome was expressed on the cell surface of S. cerevisiae by fusing with Aga2p linked to the membrane anchored protein, Aga1p. The constructed plasmid, pCTAST (7.1 kb), was introduced to S. cerevisiae EBY100 cell, and yeast transformants on YPDG plate showed the hydrolyzing activity for 4-methylumbelliferyl-sulfate and p-nitrophenyl-sulfate. When S. cerevisiae EBY100/pCTAST was grown on YPDG medium, the arylsulfatase activity of cell pellet reached about 1.2 unit/mL, whereas no extracellular arylsulfatase activity was detected. The DNA ladder in agarose prepared from agar by this recombinant arylsulfatase showed similar resolution and migration patterns with a commercial agarose. This results revealed that arylsulfatase expressed on the cell surface of S. cerevisiae could be applicable to the economic production of electrophoretic-grade agarose.

P. carageenovora 유래 arylsulfatase 유전자(astA)의 효모표면발현 plasmid pCTAST(7.1 kb)를 구축하여 S. cerevisiae EBY100에 형질전환하였고, 선별된 형질전환체들을 YPDG 배지에서 배양했을 때 4-methylumbelliferyl sulfate를 분해하여 형광을 보였다. 이는 효모에서 arylsulfatase가 활성형으로 생산되었음을 의미하였다. S. cerevisiae EBY100/pCTAST를 플라스크 배양했을 때 arylsulfatase 활성은 galactose가 완전히 소모된 배양 48시간째 최대 활성인 1.2 unit/mL로 나타났으며 배양 상등액에서는 활성이 나타나지 않았다. 효모 S. cerevisiae의 세포표면에서 발현된 재조합 arylsulfatase로 제조된 agarose를 agar와 시판용 agarose와 함께 ${\lambda}DNA$ HindIII marker를 사용하여 DNA 전기영동 성능을 비교 실험한 결과, agar보다는 재조합 arylsulfatase 처리로 제조한 agarose가 이동성이나 분리능에서 우수하였으며, 시판용 agarose와 비교하여 이동성이나 분리능이 유사한 결과를 확인할 수 있었다. 본 연구의 결과, 효모 S. cerevisiae의 세포표면에서 발현된 재조합 arylsulfatase 효소를 이용하여 한천으로부터 전기영동용 고순도 친환경적인 agarose 생물 생산 공정에 적용 가능함을 알 수 있었다.

Keywords

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