형광 Peptide를 이용한 Streptomyces griseus IFO 13350의 인산화 단백질 동정

Identification of a Protein Kinase using a FITC-labelled Synthetic Peptide in Streptomyces griseus IFO 13350

  • 허진행 (명지대학교 이과대학 생명과학과) ;
  • 정용훈 (명지대학교 이과대학 생명과학과) ;
  • 김종희 (명지대학교 이과대학 생명과학과) ;
  • 신수경 (명지대학교 이과대학 생명과학과) ;
  • 현창구 (명지대학교 이과대학 생명과학과) ;
  • 홍순광 (충북대학교 과학교육과)
  • 발행 : 2002.09.01

초록

방선균은 토양속에 서식하는 그람 양성 세균으로 세포성 장의 어느 시기에 영양세포가 이어져 연쇄상의 기균사를 형성하고 그 끝에 포자를 형성하는 동시에 생리학적 분화로 표현되는 다양한 이차대사물질을 생산한다. 이들의 복잡한 생활사에 따른 분화에는 진핵생물의 ser/thr protein kinase와 원핵생물의 his/asp acid protein kinase 등과 같은 다양한 신호전달 단백질들이 조절을 담당하고 있다. Akt kinase는 진핵생물에서 보고된 ser/thr kinase로.세포내의 다양한 신호전달기구를 조절하고 있으며, 세포내의 Akt kinase의 활성화 또는 불활성화가 세포 증식, 분화, 생존, 세포사등의 신호전달에 결정적인 역할을 담당한다. 방선균으로부터 Akt kinase와 유사한 기능을 갖는 신호전달 단백질을 규명하기 위하여, Akt kinae의 target단백질들의 인산화 부위 보존영역으로부터 나타나는 아미노산의 consensus sequence를 기초로 하여 형광물질로 라벨시킨 합성 peptide(FITC-TRRSRfESIT)를 제작하였다 제작한 기질 peptide에 인산화가 일어나면 아가 로스 전기영동상에서의 운동성에 차이가 나타나고, 이를 자외선하에서 형광 peptide를 관찰하는 방법으로 인산화 assay를 실시하였다. S. griseus IFO 13350을 배양한 cell-free extract로부터 ammonium sulfate fractionation과 DEAE-Sepharose, Mono Q, Resource Phenyl-Superose, Gel permeation 등 수 단계의 column chromatography를 통하여 Akt 유사 단백질을 정제하였다. 그 결과 방선균에도 고등생 물의 Akt와 유사한 기질특이성을 갖는 인산화 단백질이 존재하는 것으로 판단되었으며, 그 중의 하나는 분자량이 39 kDa 정도의 크기를 갖는 단백질로 판명되었다. 지금까지의 인산화 단백질 연구는 활성측정법이 어려워 연구자들에게 많은 제한을 주어 왔지만, 본 연구에서 사용한 합성 peptide를 이용하는 방법을 보다 다양한 인산화 단백질에 대하여 적용한다면, 인산화 단백질 및 조절물질 개발에 많은 도움이 될 수 있을 것으로 예상된다.

Streptomycetes is a group of Gram-positive soil bacteria that growas a branching vegetative mycelium leading to the formation of spores, and display a physiological differenti-ation related to the synthesis of many secondary metabolites including antibiotics. Their complex life cycle and multicellular differentiation require various levels of regulation and types of signal transduction systems including eukaryotic-type serine/threonine protein kinases and prokaryotic-type histidine/aspartic acid protein kinases. Akt kinase that was found in cells is a sorine/threonine kinase controlling signal pathway for multi-tude of important cellular events. The activation or inactivation of Akt kinase in the cell is one of the critical regulatory points to deliver cell proliferation, differentiation, survival or apoptosis signal. To find the regula-tory protein homologous to Akt in Streptomyces, the fluorescien-labeled synthetic peptide (FITC-TRRSR-TESIT) was designed from the consensus sequence of target proteins for Akt kinase. From the difference of the mobility between the nonphosphorylated and phosphorylated synthetic peptides on Agarose gel electro-phoresis, the Akt-phosphorylating activity was monitored. The cell-free extract prepared from Streptomyces griseus IFO 13350 and the Akt homologous protein was purified by ammonium sulfate fractionation and many steps of column chromatographies such as, DEAE-Sepharose, Mono Q, Resource Phenyl-Soporose and Gel permeation column chromatographies. As a result, the protein phosphorylating the fluorescien-labeled Akt substrate was identified and it's molecular weight was estimated as 39 kDa on SDS-PAGE.

키워드

참고문헌

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