Identification of Protein Kinases by Anti-phosphoserine/Phosphothreonine/Phosphotyrosine Antibody Immunoaffinity Column Chromatographies in Streptomyces griseus.

Protein kinases play very important role for maintaining viability in prokaryote and eukaryote. The metabolism of prokaryotic cell is generally regulated by bacterial two-component regulatory systems that are composed of histidine and asparitic acid kinases, however, some eukaryotic signal transduction system such as, serine and threonine kinases, have been also found to be involved in the regulation of morphogenesis and physiological differentiation in Streptomyces. Streptomyces griseus, a streptomycin producer, was expected to have varlous types of eukaryotic-type serine/threonine protein kinases, controlling morphogenesis. Thus, many steps of chromatographies were applied to isolate serine and threonine kinases from S. griseus IFO13350. The immunoaffinity steps using anti-phosphoserine, anti-phosphothreonine, and anti-phosphotyrosine agarose column chramatographies were successfully introduced to identify eukaryotic protein kinases from S. griseus IFO13350. Eight proteins with the expected molecular weight of 14, 29, 31, 35, 40, 52, 56, and 60 kDa, were identified on SDS-PAGE, and the their kination activity was confirmed by nonradioactive protein kination assay using FITC-labeled peptide as the substrate.

Anti-Phosphoserine/Phosphothreonine/Phesphotyrosine Antibody Immunoaffinity Column Chromatography를 이용한 Streptomyces griseus의 인산화 단백질 동정

Protein kinase는 진핵생물과 원핵생물을 포함하는 모든 생명체에서 세포생존에 절대적으로 중요한 조절 기능을 담당한다. 일반적으로 원핵생물은 histidine 과 aspartic acid kinase로 구성된 bacterial two-component regulatory system에 의하여 환경변화에 따른 유전자의 발현이 조절되지만, 방선균을 비롯한 고등 원핵생물에서는 진핵생물성의 serine/threonine kinase들이 세포분화와 같은 분화과정을 조절하고 있다. Streptomycin 생산균인 Streptomyces griseus 균주에서도 다양한 serine/threonine kinase들이 존재하는 것으로 추정되며, 이들의 기능을 밝히는 것은 생명현상을 이해하는 중요한 열쇠를 제공해 줄 것으로 기대된다. 따라서, S. griseus로부터 protein kinase 를 동정하는 연구를 실시하였으며, 기존의 복잡한 chromatography법의 단점을 보완하기 위해 anti-phosphothreonine, anti-phosphoserine, anti-phosphotyrosine antibody를 이용한 immunoaffinity column chromatography 방법을 도입하였다. 실험 결과 약 14, 29, 31, 35, 40, 52, 56, 60 kDa의 단백질을 효과적으로 동정 할 수 있었으며, nonradioactive protein kination assay 방법으로 이들의 인산화능을 확인하였다.