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Novel Approach for the Recovery of Active Recombinant Proteins from Inclusion Bodies (Ii)
|摘要:||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)。我們在執行中的99年度國科會專題研究計畫中發現，利用pH 7.0之Tris緩衝溶液可自epimerase內含體中直接溶出比活性、CD圖譜均與native epimerase相近。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。然而，利用pH 7.0 Tris buffer僅能回收內含體中約25 %的蛋白質，剩餘之蛋白質內含體勢必須要經由變性劑溶解與復性程序方能回收更多的活性蛋白質。但我們先前發現無論是利用一般稀釋、饋料稀釋、或是on-column refolding，均無法獲得理想的活性回收率。先前我們將此低活性回收率歸咎於復性過程無法獲得正確之四級結構，最近發現自內含體復性所得之epimerase與native epimerase之CD光譜有極大的差異，顯示經過重折疊之epimerase是因為未能折疊成正確的二、三級結構。Epimerase之subunit具有10個cysteine residue，雖然文獻指出native epimerase並不具有disulfide bond，但是以變性劑溶解之epimerase在冗長的溶解、復性過程中，卻有可能因氧化而產生錯誤的disulfide pairing，並進而影響其折疊成正確的二、三級結構。因此我們推論之所以無法獲得活性epimerase主要是因為disulfide bond的形成阻撓了蛋白質折疊的進行，此一論點與我們初步實驗結果吻合。因此在本計畫中我們將利用兩個多元蛋白質（N-acetyl-D-glucosamine 2-epimerase與N-carbamoyl-L-amino acid amidohydrolase）為模式蛋白，在目前執行之專題計畫之基礎上，探討在非氧化狀態下進行重折疊，以建立蛋白質包含體最佳之溶解、復性程序組合。執行本計畫所得之結果對於開發多元蛋白質之復性程序將有重大貢獻。|
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. The specific activity and the CD spectrum of the thus solubilized proteinare essentially identical to that of the native protein purified from cell lysate. However, ca. 75 % of inclusionbodies remained insoluble after prolong incubation with pH 7.0 Tris buffer. It is thus necessary to employmore vigorous conditions, such as 6 M guanidine HCL, 8 M urea, or high pH, to solubilize the proteinmolecules retained in the inclusion bodies. Unfortunately, it has been very difficult to recover activeepimerase by direct dilution, fed-batch dilution, or on-column refolding processes, presumably due to theformation of inactive, soluble oligomers. Nevertheless, based on the results obtained in the ongoing NSCresearch project, we have found that the CD spectrum of the refolded epimerase is dramatically differentfrom that of the native epimerase, suggesting the lack of activity is a result of incorrect secondary structureand thus tertiary structure rather than the failure in obtaining epimerase dimers. There are 10 cysteineresidues in each of the two polypeptide chains of epimerase. The side chains of these cysteine residues mayparticipate in the formation of disulfide binds during the folding process, although the native epimerase doesnot have disulfide bonds. We thus believe our past failures in refolding epimerase might be a result ofincorrect disulfide pairings formed during the refolding process. This is supported by our preliminaryfindings showing that activity yield can be enhanced by incorporating disulfide reshuffling systems in therefolding buffer. Based on this result, we propose to systematically investigate the feasibility of conductingprotein refolding under non-oxidative conditions in this project. Two multisubunit proteins,N-acetyl-D-glucosamine 2-epimerase and N-carbamoyl-L-amino acid amidohydrolase, will be used as themodel proteins. By carefully evaluating the specificity activity and CD spectrum of the refolded proteins, anoptimal combination of solubilization and refolding conditions will be identified. We believe the findingsmade in this study will be critical for the development of efficient refolding processes for recombinantproteins, especially for multisubunit proteins containing multiple cysteine residues.
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