Purification and Characterization of Hpa I endonuclease

Hpa I endonuclease의 정제와 특성

  • Yoon, Ho Sup (Department of Biological Science and Engineering, Korea Advanced Institute of Science and Technology) ;
  • Kang, Sun Chul (Department of Biological Science and Engineering, Korea Advanced Institute of Science and Technology) ;
  • Yoo, Ouk Joon (Department of Biological Science and Engineering, Korea Advanced Institute of Science and Technology)
  • 윤호섭 (한국과학기술원 생물공학과) ;
  • 강선철 (한국과학기술원 생물공학과) ;
  • 유욱준 (한국과학기술원 생물공학과)
  • Received : 1985.03.06
  • Published : 1985.03.01

Abstract

Hpa I endonuclease from Haemophilus parainfluenzae has been purified of homogeneity and its physical and ezymatic properties have been studied. For the purification of the enzyme, Heparin agarose, SP-sephadex C-25, DEAE-sephadex A-50 and phosphocellulose chromatography columns were used. The denatured and reduced form of the enzyme is a monomer of molecular weight of $30,000{\pm}1,000$ as judged by 10% polyacrylamide gel electrophoresis containing 0.1% sodium dodesyl sulfate. Hpa I endonuclease was maximally active at neutral pH (7.0 to 7.5) in the presence of 50 mM NaCl.

Hpa I endonuclease를 순수 정제하였다. 150g (wet weight)의 Haemophilus parainfluenzae로 부터 얻은 crude extract를 ammonium sulfate fractionation을 거친후 Heparin agarose, SP-sephadex, DEAE-sephadex, phosphocellulose 등 chromatography를 거쳐 최종적으로 0.2mg의 효소를 얻었다. Specific activity는 $2.2{\times}10^6units/mg$이었다. 얻어진 Hpa I endonuclease는 SDS-polyacrylamide gel에서 한개의 band를 보였고, 그 효소활성은 50mM NaCl과 pH 7.0과 7.5사이에서 가장 높았다.

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