介紹實驗室的研究計畫之一「TREX1的結構與功能研究及生醫應用」,解釋TREX1的催化作用及其生理功能,並說明為何TREX1功能異常會造成一系列的自體免疫疾病。此外,我們也闡述為何TREX1可以作為藥物標靶,為何其抑制劑可以應用於癌症免疫治療。We introduce one of the laboratory's research projects, " Structure-to-function studies and biomedical applications of TREX1 ", and reveal the structural basis of its catalytic properties and cellular functions. We also clarify why dysfunctional TREX1 leads to various autoimmune diseases. In addition, we explain why TREX1 can be used as a drug target and why its inhibitors can be applied in cancer immunotherapy.
Link: https://www.youtube.com/watch?v=H1pJ8PdCgLs&t=1s
本實驗室為結構生物學實驗室,利用X-ray crystallography與其他蛋白質結構決定方法(如SAXS與Cryo-EM)來解析蛋白質立體結構,以了解蛋白質結構與其生理功能的關聯性,並推導出蛋白質分子層級的作用機制。我們主要的研究標的為核酸結合蛋白質或多蛋白複合體,特別是參與在基因組穩定性維持及核酸介導免疫反應…等相關蛋白質。We are a structural biology research group and determine bio-macromolecule structures by X-ray crystallography, SAXS, and Cryo-EM. We aim to reveal the biological functions and working mechanisms of target proteins. We focus on the nucleic acid binding proteins or multi-protein complex involved in genome integrity maintenance and nucleic acid-mediated immune response.
我們利用已知的蛋白質結構為模板,透過分子接合軟體篩選出具有潛力的小分子抑制劑,再利用酵素活性分析來確認這些小分子在in vitro層級的抑制效果,並請合作實驗室進行胞內實驗來了解這些抑制劑在細胞層級之抑制活性,而通過這些實驗測試的抑制劑即具有成為小分子藥物的潛力。同時間我們也會嘗試解析出蛋白質與抑制劑之複合物晶體結構以進行小分子抑制劑的優化。 Structure-based drug design: We use protein structures and docking programs as templates and tools to find the inhibitor candidates of target proteins. These inhibitor candidates are examined by in vitro activity assays and in vivo cell-based experiments. The small molecules pass the tests are potential drug candidates. Also, we try to determine the structures of the protein-inhibitor complex for compound optimization.
實驗室已建立多種細胞外蛋白質分析技術,來量測蛋白質的活性、蛋白質的絕對分子量、蛋白質與核酸或藥物的結合能力,如Nuclease activity assay、DNA binding assay、Fluorescence anisotropy、SEC-MALS、ITC、ITF。 We have established various in vitro biochemical assays to measure the activity, absolute molecular weight, substrate/drug binding ability of target proteins, including Nuclease activity assay、DNA binding assay、Fluorescence anisotropy、Size exclusion chromatography- Multiangle light scattering (SEC-MALS)、Isothermal Titration Calorimetry (ITC)、Intrinsic Tryptophan Fluorescence (ITF).
我們實驗室在暑假時提供實驗室新進專題生與碩、博士生為期3周的實驗實作訓練課程,正式開學後老師也會利用「蛋白質表達、純化、結晶與結構解析」、「核酸結合蛋白特論」與「結構生物學」等3門課程來加深學生們研究技術相關的專業知識,理論與實作並重的教學模式可以幫助學生建構出良好的研究基礎。實驗室也會加強訓練學生的期刊閱讀、統整及簡報能力,讓學生可以成為作的好也說得好的研究人才。此外,老師會藉由小組會議的方式直接指導學生,透過與老師討論的過程,研究生不僅能構築專業的知識網絡、了解常規實驗設計邏輯、也能開拓自己的想法。
利用PCR將目標基因放大,並將insert DNA接入Plasmid DNA之中,即可完成表達載體。Amplified the target gene by PCR technique and insert the DNA into the plasmid to prepare an expression vector with a specific gene.
以TBE-Urea PAGE或 Agarose gel分析核酸受質被降解之程度,用於判斷蛋白降解核酸的效率。
We measure the nuclease activity by observing the degrading bands of DNA in TBE-Urea PAGE or agarose gel.
利用Non-denature gel 分析,以不破壞蛋白質與DNA結構的方式觀察兩者之間結合的效率。
We use Electrophoretic Mobility Shift Assay (EMSA) to measure the interaction between a protein and DNA.
使用螢光光度計(FluoroMax-4/Plus)與偏振片量測樣品螢光各向異性,並算出蛋白與受質解離常數。
We use the fluorometer (FluoroMax-4/Plus) with a polarizer to measure the fluorescence anisotropy to gain the Kd (dissociation constant).
以滴定的方式,直接偵測分子相互作用的熱量變化得到熱力學參數:結合親和力(Ka)、焓變(∆H)和結合計量(n)。可用於分析蛋白與蛋白、蛋白與受質、蛋白與藥物間的交互作用。
ITC assay is used to determine the thermodynamic parameters of interactions of the target sample in solution. It can use to analyze the interactions of the protein-protein, protein-substrate, or protein-small molecules.
當蛋白與配體結合時,會改變蛋白內色胺酸的微環境極性,導致色胺酸的螢光訊號發生衰減變化,此現象可用於分析蛋白與配體間的交互作用關係。
The binding between the protein and binding partners affects the local environment polarity around the tryptophan(s) in the protein and results in the intrinsic tryptophan fluorescence quenching. ITF can help us measure the interaction between the protein and binding partners.
我們會使用His-tag pull down以及Co-IP來量測複數蛋白質的直接交互作用關係,並會使用Far-western blot分析驗證。
We use His-tag pull-down assay and co-Immunoprecipitation (Co-IP) to detect physical interactions between two or more proteins. Far–western blot analysis is used to confirm.
SEC-MALS是一種溶液中絕對分子量測試方法。藉由Size exclusion chromatography將蛋白質樣品分離純化後再利用Multiangle light scattering測定,可以用於分析蛋白質單或多聚體形式,或蛋白質複合體的生成。
Size exclusion chromatography- Multiangle light scattering (SEC-MALS) is used to identify the sample's absolute molecular weight (M.W.) in the solution. After isolation by SEC, we can measure the M.W. of protein in monomer or polymer state and the M.W. of the protein complex.
Crosslinking assay輔以質譜分析可用於研究測試蛋白質多聚體或異質蛋白質複合體的形成,也可處理蛋白分子樣品來輔助冷凍電子顯微鏡結構測定。
Crosslinking assay combined with Mass spectrometry can measure the formation of protein-polymer or hetero-protein complex. It is also helpful for treating the protein sample for facilitating the structure determination by cryogenic electron microscopy (Cryo-EM).
記錄當溫度上升時,蛋白質疏水性區域因變性而暴露於外並與螢光染劑產生交互作用時的訊號變化,用於檢測蛋白質在不同實驗條件下的結構穩定程度,如不同成分的緩衝溶液、受質或配體,亦可觀察蛋白質突變對自身結構穩定度的影響。
When the temperature rises, the hydrophobic region of the protein is exposed due
to denaturation, and the signal changes when it interacts with the fluorescent dye.
TSA is used to detect the structural stability of the protein under different
experimental conditions, such as under various buffer solutions, components,
substrates, or ligands. The protein mutation on the inherent structural stability of
the protein can also be observed.
在人的體內,有一種神祕的基因「CCR5(C-C chemokine receptor type 5)」。它所轉譯出的蛋白質是一種由數個transmembrane helix所組成且...
+ READ MORERobert C. Robinson教授與Caner Akil博士使用台灣光子源05A蛋白質結晶學實驗設施,研究Asgard古菌的基因體,研究顯示Asgard古菌具有功能性的「肌動...
+ READ MORE蜘蛛絲是一種由蜘蛛分泌的蛋白質纖維,用來建構蜘蛛網以捕捉獵物和孕育子代的場所,也能提供在樹間行走的橋樑,久未整理的倉庫時常會佈滿蜘蛛絲,清理起來十分的惱人,但你知道那撥起來柔軟的蜘...
+ READ MORE