製造及純化可溶性重組COQ5蛋白之方法及其製得的可溶性重組COQ5蛋白METHOD OF PRODUCING AND PURIFYING SOLUBLE RECOMBINANT COQ5 PROTEIN AND SOLUBLE RECOMBINANT COQ5 PROTEIN THEREOF

專利類型

發明

專利國別 (專利申請國家)

美國

專利申請案號

13/600,963

專利證號

US 8,907,062 B2

專利獲證名稱

製造及純化可溶性重組COQ5蛋白之方法及其製得的可溶性重組COQ5蛋白METHOD OF PRODUCING AND PURIFYING SOLUBLE RECOMBINANT COQ5 PROTEIN AND SOLUBLE RECOMBINANT COQ5 PROTEIN THEREOF

專利所屬機關 (申請機關)

長庚大學

獲證日期

2014/12/09

技術說明

合成輔酶Q所需之酵母菌COQ5蛋白已被鑑定出，而且是一個粒線體蛋白。為了研究人類COQ5蛋白，我們想要純化有His標記之人類COQ5重組蛋白，但是其卻在E. coli內形成內涵體而無法在無尿素的任何條件下被純化出來。本發明係關於一種在非變性條件下，製造及純化以可溶性形式表現於大腸桿菌之重組COQ5蛋白的方法。本發明之製造及純化方法特徵係在於，預先以低濃度離子性清潔劑處理細菌之溶菌產物，且純化過程中無使用尿素，而能避免使純化出之蛋白會有聚集或沉澱現象的。我們因而能取得足量可溶性重組蛋白來製作親和性管柱所純化之兔子多株抗體。西方墨點法之結果顯示我們所製造出之多株抗體能專一地偵測到人類細胞中內生性COQ5蛋白之全長和預測之成熟型式。 Yeast COQ5 protein for biosynthesis of coenzyme Q has been identified and was a mitochondrial protein. We intended to purify His-tagged recombinant human COQ5 protein, but it formed inclusion body in E. coli and could not be purified without urea under all conditions tested. The present invention relates to a producing and purifying method of soluble recombinant COQ5 protein, which is expressed in soluble form by E. coli, under native conditions. The method is characterized by pre-treating bacterial lysate with low concentration of ionic detergent before purification; and the purifying method without the use of urea to avoid the problems of aggregation or precipitation of purified protein. We therefore could have sufficient amount of soluble recombinant protein to generate affinity column-purified rabbit polyclonal antibody. Results of Western blot showed that both full-length form and predicted mature form could be specifically recognized by the polyclonal antibody we produced.

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