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The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Cloning and Expression of Inositol Monophosphatase Gene from Streptomyces coelicolor A[3]2

Streptomyces coelicolor A[3]2에서 Mycothiol 생합성에 관여하는 Inositol Monophosphatase 유전자의 클로닝 및 발현

  • Kim Jin Kwon (Department of Environmental Engineering, Chosun University) ;
  • Choi Hack Sun (Department of Environmental Engineering, Chosun University) ;
  • Kim Seong-Jun (Department of Environmental Engineering, Chonnam National University) ;
  • Kim Si Wouk (Department of Environmental Engineering, Chosun University)
  • 김진권 (조선대학교 공과대학 환경공학부) ;
  • 최학선 (조선대학교 공과대학 환경공학부) ;
  • 김성준 (전남대학교 공과대학 환경공학과) ;
  • 김시욱 (조선대학교 공과대학 환경공학부)
  • Published : 2004.12.01
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Abstract

Mycothiol (MSH), a low molecular antioxidant thiol compound, was purified and analyzed from Streptomyns coelicolor A[3]2 by the monobromobimane fluorescence detection method modified by this lab. Through HPLC chromatpgram, MSH fraction was obtained following the elution time of standard MSH (donated by Dr. Robert C. Fahey). That MSH showed the highest concentration among the thiol compounds contained in the cell indicated that MSH was the key thiol compound having antioxidant activity. To understand the role of gene of inositol monophosphatase (I-1-Pase) involved in the MSH biosynthesis, it was isolated from S. coelicolor A(3)2 and cloned and overexpressed in the Escherichia coli. The expressed I-1-Pase was purified through Ni-NTA column. The soluble protein consisted of 281 amino acids, and the molecular weight was 32 kDa. I-1-Pase of S. coelicolor A(3)2 had the sequence homology with those of human and E. coli by 24 and $25\%$, respectively, and had two conserved domains (mofif A and motif B) which were typical of I-1-Pase.

S. coelicolor A3(2)로부터 항산화 저분자 thiol분자인 MSH를 HPLC 및 monobromobimane 형광 검출 방법으로 분리${\cdot}$정제하여 그 존재를 확인하였다. 표준물질인 MSH-bimane과 동일하게 용출되는 MSH 분획을 확인하였으며 여러 thiol 분획 중 MSH 분획이 가장 많은 것으로 보아 MSH가 S. coelicolor의 주된 thiol 화합물로 판단되었다. MSH 생합성에 관여하는 효소 중 I-1-Pase의 유전자의 기능을 알아보기 위하여 이 유전자를 방선균에서 분리한 후 대장균에 클로닝하여 과도발현시켰다. 발현된 I-1-Pase를 Ni-NTA column을 사용하여 정제하였다. 정제된 I-1-Pase는 soluble protein으로 281개 아미노산으로 구성되어 있으며 분자량은 32 kDa이었다. 인간 및 대장균의 I-1-Pase와 각각 24와 $25\%$의 sequence homology를 보였으며, 기존의 I-1-Pase가 가지고 있는 공통의 I-1-Pase motif A와 motif B를 S. coelicolor A3(2)도 가지고 있는 것으로 확인되었다.

Keywords

References

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