大腸桿菌中含硫醇基之蛋白質 之蛋白質體學

Abstract

隨著後基因體時期的來臨，為了進一步了解生物系統的運作，所以將研究重點放在蛋白質體學上。為了達到不同的研究目標，發展出許多關於蛋白質體學的實驗方法。本篇論文中使用activated-thiol sepharose 4B 當作共價層析管柱，並以此為工具研究大腸桿菌蛋白質體。首先，我們以牛胰臟去氧核糖核酸水解酶 (bpDNase) 及其突變株 (brDNase C101A) 測試此共價層析法之性質。由於bpDNase 具有兩對雙硫鍵且缺乏單一硫醇基，因此無法接合在膠體上。反之，brDNase C101A 具有一個單獨的硫醇基(Cys104)，可以成功地被連接於共價層析膠體上，且用25mM DTT沖提下來後仍具有酵素活性。接著我們以共價層析法分析大腸桿菌 (E.coli ) 的蛋白質體，將沖提下來的蛋白質以二維電泳分離，再用電噴灑串聯式質譜儀(ESI-LC-tandem mass spectrometry) 鑑定蛋白質身分。在二維電泳膠片上共有四十三個蛋白質被鑑定出來，其中包括九個氫化酵素 (hydrogenase)、四個氧化還原相關酵素 (redox-related enzymes)、兩個熱休克性蛋白 (heat-shock proteins) 及四個核糖體組成蛋白 (ribosomal proteins) 等。若E.coli 遭受轉型作用的刺激，我們發現有β-galactosidase, tryptophanase and chloramphenicol acetyl transferase 會大量表現。為了觀察E.coli 在高度氧化環境下的蛋白質體變化，我們在培養液中加入5 mM H2O2，經由共價層析法分析其蛋白質體後，發現alkyl hydroperoxide reductase (AhpC) 有大量表現。根據前述結果，發現此共價層析管柱可能與含金屬離子之蛋白質有較強結合能力，因此我們在E.coli 的蛋白質混合液中加入20mM EDTA 再通入共價層析管柱中，結果發現EF-Tu 有大量增加。根據這些實驗結果，我們提出利用共價層析法，再結合二維電泳及液相層析質譜儀，應可有效且有系統性地分析蛋白質體中含有高度反應性硫醇基的蛋白質。

In post-genomic era, the field of proteomics has emerged for the analysis of biological systems. Some proteomic approaches were developed to achieve different research goals. In this paper we used the activated-thiol sepharose 4B as a covalent chromatographic beads to study E.coli proteome. We examined the feasibility of activated-thiol sepharose 4B using native bpDNase and brDNase (C101A). As expected, the bpDNase with two pairs of disulfide bonds and no free sulfhydryl group can not be bind to the activated-thiol sepharose 4B beads, while brDNase (C101A), having one free sulfhydryl group on Cys104, could be linked to the activated-thiol sepharose 4B beads and eluted in its fully active form by 25mM DTT. Then, E.coli lysate was applied to the covalent chromatography and the eluent was further analyzed by two dimensional electrophoresis (2DE) and ESI-LC-tandem mass spectrometry. Forty-three proteins on the 2DE PAGE were identified. Among them, there were 9 hydrogenase enzymes, 4 redox-related enzymes, 2 heat-shock proteins and 4 ribosomal proteins. If the E.coli was challenged with transformation, β-galactosidase, tryptophanase and chloramphenicol acetyl transferase were found to be overexpressed. We also found that when E.coli was under H2O2 treatment, the expression of alkyl hydroperoxide reductase (AhpC) enzyme was increased. Pre-treatment of E.coli lysate with 20mM EDTA resulted in a large increase amount of EF-Tu in the eluents. Covalent chromatography and 2D eletrophoresis/tandem mass spectrometry were employed to analyze the E.coli proteome, and the reactive sulfhydryl-containing proteins could be concentrated. With these approaches, it would be possible to systematically investigate the alteration of redox proteomics under challenges in E.coli.