建構羊毛硫肽之融合蛋白質以進行膦位點特異性標記

Abstract

蛋白質標記常用於探討蛋白質功能與生物化學活性，例如科學家常以螢光分子標誌蛋白質以觀察該蛋白質在生物體內這類複雜環境的移動及定位。自體發螢光蛋白標籤、HaloTag標籤及利用點擊化學的標籤被發展來可視化蛋白質之位置。前人發現，膦能夠選擇性地與胜肽鏈上之α,β-不飽和胺基酸反應，且沒有發現伴隨副反應。天然物中常帶有α,β-不飽和胺基酸的結構，特別是在羊毛硫肽（lanthipeptides）這類天然物。 羊毛硫肽是一類核醣體合成及轉錄後修飾胜肽（ribosomally synthesized and post-translationally modified peptides, RiPPs），它們被編碼進DNA，在轉錄及轉譯後，相關的酵素在其身上進行修飾。這類胜肽在結構上帶有羊毛硫胺酸以及α,β-不飽和胺基酸，如去氫丙胺酸（Dha）和去氫丁胺酸（Dhb），他們是從絲胺酸、蘇胺酸與半胱胺酸經過脫水及環化而成。負責催化這類反應的酵素可以是一整個蛋白質，亦可以是個別蛋白質分工催化。Cacaoidin是一個近年被發現的羊毛硫肽，它的前驅物經過脫水反應後帶有 8 個α,β-不飽和胺基酸，它的生物合成基因簇（biosynthetic gene cluster, BGC）含有獨立的激酶與解離酶，這兩個酵素共同構成脫水複合物。 在這篇研究中，我們計劃把cacaoidin前驅物胜肽與蛋白質融合，將其與融合的脫水複合物酵素共同表達，以發展一套膦位點特異性標記系統。我們已經透過基質輔助雷射脫附電離—飛行時間質譜儀確認融合蛋白上的前驅物胜肽確實有進行脫水反應產生α,β-不飽和胺基酸，以及使用蛋白質電泳與液相層析質譜確認膦成功標記在融合蛋白上。

Understanding protein function and biochemical activity often requires the help of protein labeling. For example, fluorescent tags on proteins help scientists track the position and transportation of proteins in a complicated surrounding. Autofluorescent-protein tags, HaloTag and labeling via click reaction were developed to visualize these processes. However, these tags often have a large molecular weight or require time-consuming steps to build the system. It has been reported that phosphines selectively react with α,β-unsaturated residues on peptides with no detectable side reaction. α,β-unsaturated residues are abundant in natural products, especially lanthipeptides. Lanthipeptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs), encoded in DNA and modified by various enzymes. They were characterized by lanthionine and α,β-unsaturated residues, such as dehydroalanine (Dha) and dehydrobutyrine (Dhb), which are derived from cysteine, serine or threonine residues via enzyme-catalyzed dehydration followed by cyclization. The enzymes responsible for dehydration and cyclization can be one whole large protein or 2 to 3 individual proteins. The precursor of a recently discovered lanthipeptide—cacaoidin—contains 8 α,β-unsaturated residues after dehydration. Its biosynthetic gene cluster (BGC) encodes individual kinase and lyase, which form a dehydration complex together. In this study, we aim to fuse the cacaoidin precursor peptide to a protein and co-express it with the dehydration enzyme in order to develop a system for phosphine-mediated site-specific protein labeling. Dehydration of the precursor peptide has been confirmed by MALDI-TOF MS, and successful phosphine labeling of the fusion protein was confirmed by SDS-PAGE and LC-ESI MS.