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Novel Approach for the Recovery of Active Recombinant Proteins from Inclusion Bodies
利用大腸桿菌大量表現重組蛋白質往往在細胞質中或週質中形成不溶於水、不具生物活性的蛋白質聚集體(或稱蛋白質內含體)。由於蛋白質內含體可利用簡單的離心及清洗步驟加以純化，因此如能發展一自內含體回收活性蛋白質的高效率的製程，則內含體的形成，反而提供一個有效純化重組蛋白質的捷徑。本實驗室最近發現，利用pH 7.0 之Tris 緩衝溶液可自內含體中直接溶解出比活性、CD 圖譜均與native form 相近之蛋白質，且其回收率遠高於自粗酵素液純化所得之native protein。此一發現與文獻認為內含體係由folding-prone polypeptide chain 組成及細胞內內含體形成具可逆性之理論不謀而合。因此，本計畫擬以三年的時間以epimerase、carbamoylase、racemase 進行系統化之研究：第一年我們將著重在建立自此三種內含體直接以Tris 緩衝溶液回收活性蛋白質之最適化條件，並以CD 分析其結構差異。第二年我們將探討誘導後培養溫度、培養基添加劑對內含體形成率及自該內含體回收活性蛋白質效率之影響，從而建立其與內含體結構與組成之影響。由於直接以pH 7.0 或8.0 之Tris 緩衝溶液自內含體回收活性蛋白質後，尚有約60%之未溶解內含體，因此有必要進一步變性劑溶解、復性以取得更多之活性蛋白質，因此在第三計畫年度我們將著重在以梯度透析、饋料稀釋、管柱復性程序回收殘餘內含體之活性蛋白。執行本計畫所獲致之研究成果，將對較難復性之multisubunit protein內含體，建立一可行之活性蛋白回收製程，並可瞭解發酵環境、添加劑、蛋白質結構對內含體之形成與其結構組成之影響，此一知識除了可做為蛋白質重摺疊最適化技術之指引外，更能提供in vivo proteinfolding/refolding 之學術論述基礎，兼具產業應用與學術發展價值。
The advances in recombinant DNA technology have made possible the large scale production of manytherapeutic proteins. However, the over-expression of recombinant proteins in E. coli usually leads to theformation of insoluble, intracellular protein aggregates, or inclusion bodies. Since inclusion bodies can beeasily separated from soluble proteins and cell debris by centrifugation, the formation of inclusion bodiesmay provide an alternative route for the purification of recombinant proteins, provided with an efficientstrategy for the refolding of proteins from inclusion bodies. We have recently found that it is possible torecover active protein from inclusion bodies with pH 7.0 Tris buffer without prior unfolding procedure. Thisfinding is consistent with the reported hypothesis suggesting that inclusion bodies are consisting offolding-prone polypeptide chains. Based on this finding, we propose in the project to conduct a systematicstudy aiming at establishing an efficient protocol for the recover and elucidating the effects of fermentationconditions, additives, and protein structure on the composition of inclusion bodies. Three multisubunitenzymes, epimerase, carbamoylase, and racemase, will be used in this three-year project as the modelproteins. In the first project year, we will focus on finding the ideal pH for the recovery of active, solubleenzymes form these inclusion bodies and comparing the structure of the recovered proteins with that ofnative proteins by circular dichroism. In the second project year, we will perform a systematic study aimingat identifying the effects of additives, such as sucrose, in the fermentation medium and the post-inductionincubation temperature on the structure of inclusion bodies. This will be done by comparing the efficiency ofactive protein recovery with Tris buffer and the solubility of inclusion bodies in various denaturants. Finally,in the third project year, we will engage in developing efficient processes for the refolding of proteins fromthe residual inclusion bodies obtained in the first and second project years. This will be achieved by firstidentifying the ideal protocol for the solubilization of inclusion bodies obtained at different fermentationconditions. The solubilized proteins will then be refolded by gradient dialysis, fed-batch dilution, onon-column refolding processes. By compiling the results obtained in this study, we will be able to develop aefficient strategy for he recovery of active proteins from inclusion bodies, establish the correlations betweenprotein structure/post-induction incubation temperature/additives and the composition/structure of inclusionbodies. The strategy thus developed will be useful for setting a guideline for the recovery of active proteinson industrial scales; the knowledge acquired will contribute to the elucidation of the underlying mechanismsof protein folding and unfolding in vivo in bacteria.
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