생명과학회지 (Journal of Life Science)

  • 제22권3호
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  • Pages.306-311
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  • 2012
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
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Pseudomonas mandelii의 lipase 유전자 클로닝, 발현 및 정제

Cloning, Expression, and Purification of a Lipase from Psychrotrophic Pseudomonas mandelii

  • 김준성 (대구대학교 의생명과학과) ;
  • 이창우 (대구대학교 의생명과학과)
  • 투고 : 2012.02.08
  • 심사 : 2012.03.13
  • 발행 : 2012.03.30
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초록

내냉성 세균인 Pseudomonas mandelii로부터 lipase 유전자(lipT)를 클로닝하고 염기서열을 분석하였다. 열린해독틀 (open reading frame)은 1,686 bp로 구성되어 있고, 562개의 아미노산을 코딩한다. 서열분석 결과 많은 세린 효소에서 발견되는 Gly-X-Ser-X-Gly 모티프가 존재한다(Gly-His-Ser-Leu-Gly). 재조합 LipT 단백질은 대장균에서 주로 inclusion body 형태로 발현되었다. 니켈 친화성 크로마토그라피 방법으로 LipT 단백질을 분리하였으며 소량의 LipT 단백질이 refold 되었다. 이 효소는 p-nitrophenyl butyrate (C4)과 p-nitrophenyl octanoate (C8)에 대해 기질 특이성을 나타내었다.

A gene encoding a lipase, lipT, was cloned from the psychrotrophic bacterium Pseudomonas mandelii and sequenced. An open reading frame of 1,686 bp was found that encodes a polypeptide consisting of 562 amino acid residues. Sequence analysis revealed a Gly-His-Ser-Leu-Gly sequence, which matches the consensus Gly-X-Ser-X-Gly motif conserved among lipolytic enzymes. The recombinant LipT protein was predominantly expressed as inclusion bodies in Escherichia coli and subsequently purified by nickel-chelate affinity chromatography. A small fraction of LipT was refolded, and the subsequent LipT exhibited substrate preferences for p-nitrophenyl butyrate (C4) and p-nitrophenyl octanoate (C8).

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