생명과학회지 (Journal of Life Science)

  • 제14권5호
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  • Pages.840-846
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  • 2004
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
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Saccharomyces cerevisae에서 한국산 겨우살이 유래 lectin A 및 B 유전자의 발현

Expression of Recombinant Korean Mistletoe(KM) Lectin and B genes in Saccharomyces cerevisiae

  • 최윤혁 (한동대학교 생명공학연구소) ;
  • 김종배 (한동대학교 생명공학연구소) ;
  • 양웅석 ((주)미술 바이오텍) ;
  • 황철원 (한동대학교 생명공학연구소)
  • 발행 : 2004.10.01
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초록

본 연구는 한국산 겨우살이 lectin유전자 (A 및 B chain) 을 효모 (Saccharmyces cerevisiae) 에 형질 전환시키는 시스템을 사용한 것으로, 효모내 효과적인 lectin유전자 발현을 위하여 유전자 상에 Kozak translation initiation sequence를 PCR을 이용 삽입, 변형시켜 재 클로닝 하였다. 변형된 lectin A 및 B 유전자를 포함하는 재조합 플라스미드는 S. cerevisiae INVSc (MATa, his3 $\Delta$l, leu2, trpl-289, ura3-52) 에 형질전환 되었다. 형질전환된 효모는 ABI 3700 system을 이용한 DNA 염기서열 분석을 통해 확인되었고 재조합 한국산겨우살이 lectin 발현을 위해 2% galactose를 사용하여 유도발현되었다. 재조합 lectin A 및 B 단백질은 SDS-PACE 및 western blotting 분석을 수행한 결과 약 29kDa 크기로 확인되었다. 재조합 lectin은 세포내 가용성 단백질 1mg중 1.24∼l.75 $\mu\textrm{g}$ 수준으로 발현되어짐을 ELISA 분석을 통해 확인하였다. 한편 lectin 유전자는 galartose 유도발현 후 36시간이 되 었을 때 발현량이 최대가 되었으며 lectin A 유전자의 경우 48 시간 이후에는 발현이 억제되었다.

A study for expression of Korean Mistletoe (KM) lectin gene (A,B) in Saccharomyces cerevisiae was done using transforming system of yeast. In order to overexpress the genes efficiently in yeast, two lectin genes (A,B) were re-cloned and modified including Kozak translation initiation sequence using PCR amplification. The constructed plasmids containing modified lectin A and B genes were transformed to S. cerevisea INVSc (MAT G, his3 $\Delta$1, leu2, trpl-289, ura3-52). The transformed cells were identified by DNA sequencing with ABI3700 system and induced with 2% of galactose for recombinant KM lectin (rKM lectin) protein. The rKM lectin A and B proteins were determinated about 29kDa size of protein by SOS-P AGE and western blotting analysis. The expressed recombinant lectin was determinated 1.24∼1.75 $\mu\textrm{g}$ per 1 mg of cytosolic soluble protein by sandwich ELISA method. Moreover the lectin genes were expressed as maximum level at 36 h after galactose induction and lectin A gene was were repressed after 48 h.

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