親和純化啤酒酵母菌粒線體之定量研究

Affinity Purification of Mitochondria from the Yeast Saccharomyces cerevisiae：A Quantitative Study

鍾欣彧(Xin-Yu Chung)

指導教授 ： 陳銘凱

淡江大學/理學院/化學學系碩士班/碩士(2022年)

https://doi.org/10.6846/TKU.2022.00461

摘要

現今已出現多種因粒線體缺失導致的疾病，透過粒線體治療可使患者的病情穩定許多。治療用粒線體的傳統純化方式是以不同轉速，經多次離心的方式取得，但缺點不僅費時，取得粒線體的數量稀少且形態偏差。已有更好的方法取代傳統純化粒線體－串聯親和純化。
而本實驗選用純化蛋白質的方法—串聯親和純化（Tandem Affinity Purification，TAP）來純化酵母菌的粒線體。在開始純化之前，為了解決因核酸堵塞管柱的問題，加入核酸水解酶（Benzonase）。粒線體外膜蛋白上具 C 端修飾的 TAP tag－Calmodulin Binding Protein（CBP）將與被鈣離子改變構型過後的 Calmodulin Sepharose 4B 結合。注入兩種鹽分濃度不同的清洗液清洗管柱後，加入可螯合金屬的 EGTA（Ethylene glycol tetraacetic acid），讓粒線體隨著鈣離子沖提出管柱。
利用粒線體獨有的琥珀酸脫氫酶（Succinate Dehydrogenase，SDH）做為檢測粒線體數量的依據，其轉換琥珀酸（Succinate）為富馬酸（Fumarate），並放出電子。靛色的 2,6-二氯靛酚（Dichloroindophenol, DCIP），接收到電子後會變為無色，分光光度計（ChromTech, UV-3100）可檢測此反應。在波長為 600 nm下，吸收值隨著時間由高至低，並由每分鐘 DCIP 減少的濃度當作粒線體的活性，以推測粒線體的產率。
為了讓數據得到驗證，在沖提液中加入 MitoTracker Green FM 染色粒線體後，使用顯微鏡觀察其形態有完整保留且沒有受到破壞，證實酵母菌粒線體經由串聯親和純化是可行的。
由以上結果可知，串聯親和純化酵母菌的粒線體為可行的實驗方法，並期望往後實驗可以延伸應用至人體細胞粒線體的純化，讓粒線體治療能夠更方便。

關鍵字

粒線體 ； 串聯親和純化 ； CBP ； SDH ； MitoTracker Green FM

並列摘要

Nowadays, there are a variety of diseases caused by defects of mitochondria. These diseases can be stabilized a lot by mitochondrial replacement therapy. Most of the mitochondria used in the treatment are obtained by conventional purification, where mitochondrial purification is processed by multiple steps of centrifugation. However, the loss rate of this method is extremely high that a lot of mitochondria cannot be reserved and their types are also biased. There is a better method to replace the conventional purification of mitochondria – Tandem Affinity Purification(TAP).
In order to ameliorate this situation, this experiment utilizes a method derived from TAP. Before the purification beginning, nucleic acid hydrolase－Benzonase was added to hydrate nucleic acid clogging the column. The TAP tag with a C-terminal modification on the mitochondria outer membrane protein will bind to Calmodulin Sepharose 4B after its configuration has been changed by calcium ions. After washing the column to remove contaminants with a different salt concentration of wash buffers, EGTA, which can chelate metals, is added to make mitochondria elute out of the column along with calcium ions.
Succinate dehydrogenase (SDH) is a mitochondrial unique enzyme that is used as the basis for the detection of mitochondrial concentration, which converts succinate to fumarate and emits electrons. The indigo 2,6-dichloroindophenol (DCIP) becomes colorless due to receiving electrons from the succinate-fumarate reaction, and this change can be detected by a spectrophotometer (ChromTech, UV-3100). At a wavelength of 600 nm, absorbance will be decreased as time goes on, and the decreasing concentration of DCIP per minute was used as a mitochondrial activity to estimate mitochondrial yield.
In addition, in order to verify mitochondria exist in the eluted products, I added MitoTracker Green FM to the eluent. The morphology of mitochondria was observed by microscopy to be intact and not damaged, confirming that yeast mitochondrial tandem affinity purification is feasible.
Based on experimental results, tandem affinity purification of the mitochondria can be proved feasible. Eventually, I hope this experiment method can be applied to the purification of mitochondria of human cells so that mitochondrial treatment can be more convenient.

並列關鍵字

Mitochondria ； Tandem affinity purification ； CBP ； SDH ； MitoTracker Green FM

參考文獻

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