Yeast내에서 MEK1 융합 단백질 발현 및 Lethal Factor 활성 검증 = Expression of MEK1 Fusion Protein in Yeast for Developing Cell Based Assay System, a Major Substrate of LeTx

국문 초록 (Abstract)

Anthrax lethal toxin은 탄저병의 치사원인이 되는 독소이며, Lethal toxin은 두 종류의 단백질 PA (Protective antigen)과 LF (lethal factor)로 구성되어 진다. PA는 세포표면의 수용체와 결합하여 LF를 세포질 안으로 이동시켜 주는 역할을 한다. LF는 금속 이온$(Zn^{2+})$ 의존적 단백질 가수분해 효소로써 MKKs[MAPK (mitogen-activated protein kinase) kinases] 집단 단백질의 아미노 말단 부분을 절단하여 대상 세포를 죽음으로 유도하는 것으로 알려져 있다. 본 연구에서는 LF에 대한 특성 분석 및 억제제 개발에 과한 연구를 위해 cell-based high-throughtput screens 개발에 선행되어야 하는 기초 자료를 마련하는데 그 목적이 있다. 이를 위하여 LF의 절단 대상이 되는 기질이 MEK1을 yeast내에서 동시 발현시켜 LF의 활성을 검증하였다. 먼저 효모(Saccharomyces cerevisiae)를 숙주로 하여 LF의 기질인 MEK1 발현 vector를 구축하였고, 구축된 발현 system을 기본을 LF 활성을 검증하고자 yeast에 형질전환하여 plasmid의 안전성 및 MEK1 유전자의 발현 및 LF에 의한 MEK1 아미노말단의 절단 부위를 확인하였다. 본 연구는 세포내 검증 system 도입의 기초적 자료를 제공하였으며, yeast내의 MEK1 발현은 탄저병의 저해제 선별 및 활성 측정 검증을 생체에서 고효율적이며, 안정적으로 할 수 있다는 가능성을 나타냈다.

다국어 초록 (Multilingual Abstract)

Lethal toxin is a critical virulence factor of anthrax. It is composed two protein: protective antigen (PA) and lethal factor (LF). PA binds to specific cell surface receptors and, forms a membrane channel that mediates entry of LF into the cell. LF is a zinc-dependent metalloprotease, which cleaves MKKs [MAPK (mitogen-activated protein kinase) kinases] at peptide bonds very close to their N-termini. In this study, we suggest application of cell-based assays in the early phase of drug discovery, with a particular focus on the use of yeast cells. We constructed MEK1 expression system in yeast to determine LF activity and approached cell-based assay system to screen inhibitors, in which the results covering the construction of LF-substrate in yeast expression vector, expression, and LF-mediated proteolysis of substrate were described. These results could provided the basic steps in design of cell-based assay system with the high efficiency, rapidly and easy way to screening of inhibitors.

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