高效率的重組Tau蛋白純化方法與其聚集特性之研究

A facile method for recombinant Tau protein purification and a study on its aggregation properties

吳穎茹(Ying-Ju Wu)

指導教授 ： 陳振中

國立臺灣大學/理學院/化學研究所/碩士(2022年)

https://doi.org/10.6342/NTU202203913

摘要

Tau蛋白病（Tauopahty）涵蓋多種神經退化性疾病，例如阿茲海默症（Alzheimer’s disease）、匹克症（Pick’s disease, PiD）、皮質基底核退化症（Corticobasal degeneration, CBD）等，臨床上會表現出一系列認知能力缺失和運動行為障礙等症狀，而Tau蛋白聚集形成的絲狀纖維糾結（neurofibrillary tangles, NFTs）是造成神經退化性病變的關鍵，也是Tauopahty的標誌性特徵，原先可溶性的Tau蛋白負責維持軸突微管的結構與穩定性，在發生聚集後，形成不可溶的纖維狀Tau蛋白聚集體，堆積在神經元或神經膠質，進而影響到神經的功能與活性。為了治療這類型疾病，科學家需要了解Tau蛋白的生理功能與其聚集體引發的致病機制，而在體外有效地純化出高質量的重組Tau蛋白是進行研究必不可少的首要步驟，且須確保所獲得的Tau蛋白具有活性。
本研究主要是設計一套製備的方法，來表達和純化未被標記的全長Tau蛋白（2N4R）。藉由大腸桿菌的T7啟動子系統以IPTG誘導進行表達後，在沸水中加熱與超音波震盪的破菌方法能達到初步純化的效果，接著以陰離子交換層析法和粒徑排阻層析法進行純化，收集液通過蛋白質膠體電泳確認Tau蛋白流出管柱的時間，並以銀染與質譜鑑定純度，依據上面簡述之方法，從1L LB培養液中的大腸桿菌中可製備出10 mg的可溶性重組Tau蛋白，純度為95%以上。
利用花生四烯酸和聚尿苷酸進行誘導，測試製備得到Tau蛋白的聚集特性，Tau蛋白聚集體形成的β 折疊結構由硫磺素T螢光探測法進行測定，具有良好的再現性，這能證明我們製備的2N4R Tau蛋白適用於後續聚集體特性之研究。

關鍵字

Tau蛋白 ； 純化 ； 花生四烯酸 ； 聚尿苷酸 ； ThT染色螢光探測法

並列摘要

Tauopathies represent a class of neurodegenerative disorders, including Alzheimer’s disease, Pick’s disease, and corticobasal degeneration. They usually exhibit a series of symptoms that include cognitive deficits as well as movement disorders. Meanwhile, neurofibrillary tangles (NFTs) formed by tau aggregations are not only recognized as the hallmark characteristics of tauopathies but also key toxic agents causing neurodegenerative diseases. The original function of soluble tau is responsible for maintaining the structure and stability of axonal microtubules. Once forming and accumulating in neurons or glial cells, insoluble fibrillar aggregates negatively impact the function and activity of the brain. There is no known cure for these disorders. Scientists are still trying to understand the pathogenic mechanisms related to its aggregations. Because the study of aggregation processes is very sensitive to any impurities, efficient purification of high-quality recombinant tau protein in vitro is an essential first step.
Although it is customary to express recombinant Tau protein in E. coli, the yield and purity are usually suboptimal. In this work, a facile protocol has been devised for the efficient production of untagged and full-length Tau protein (2N4R). The expression system was based on the T7 promoter system in E. coli for IPTG-induced expression. Bacterial cell wall was disrupted by not only sonication but also direct boiling for 10 minutes, achieving the effect of preliminary purification. Tau protein remained in the heat-soluble fraction, and was further purified using anion exchange chromatography and size-exclusion chromatography. The purity was assessed with silver staining after SDS-PAGE and mass spectrometry. The typical yield was 10 mg of Tau protein from 1 L of E. coli culture, with a more than 95%, without any visible contaminants by silver stain.
The aggregation property of this Tau protein preparation was tested under induction conditions with arachidonic acid (AA) and polyuridylic acid (polyU RNA). The formation of cross-beta structures was detected by thioflavin T fluorescence, with good reproducibility, which confirmed that our Tau protein preparation is suitable for the study of aggregation properties.

並列關鍵字

Tau ； purification ； arachidonic acid ； polyuridylic acid ； ThT fluorescence assay

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