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標題: TorD與DmsD對雙精胺酸轉位路徑訊息序列交叉活性之研究
A Systematic Study on the Cross Activities of DmsD and TorD on Potential Tat Signal Peptides
作者: 林松池
關鍵字: 基礎研究;twin-arginine;化學工程類;雙精胺酸;大腸桿菌;細胞間質;轉位;E;coli;periplasm;translocation
Since the secretion of recombinant proteins into the periplasm of E. coli cansignificantly increase the yield of active, authentic recombinant proteins due to thephysiological characteristics associated with the periplasm and the secretion process per se, itis often desirable to transport recombinant proteins into the periplasm of E. coli. The secretionof proteins into the periplasm is generally achieved via the well-studied Sec pathway.However, since Sec pathway can only transport proteins in an unfolded state, it cannot beexploited for the translocation of proteins that contain complex cofactors, require delicatedisulfide pairing, or have complex tertiary or quaternary structures for biological functions.An alternative ATP-independent pathway, twin-arginine translocation (Tat) pathway, hasrecently been discovered. The translocation of recombinant proteins via the Tat pathway hasattracted enormous interest because of its ability to transport cofactor-containing proteins infolded state. However, the translocation efficiency via the Tat pathway is in general muchlower than that via the Sec pathway. In our previous study we have shown that theco-expression of a molecular chaperone TorD can promote translocation via the Tat pathway.We have further demonstrated in our recent study that co-expression of DmsD has a similareffect on protein translocation and the presence of cross activity between TorD and DmsD.Results of our preliminary study further show that co-expression of TorD can enhance thetranslocation of green fluorescence protein (GFP) fused with the seemingly unrelated signalpeptide of TorZ, but not DmsD. In light of these findings, we propose to conduct a systematicstudy aiming to elucidate the effect of DmsD and TorD co-expression on the translocation ofGFP with 29 putative signal peptides with characteristic Tat signal peptide motif in E. coli. Invitro binding experiments will also be carried out to elucidate the underlying mechanism forenhanced protein translocation via the Tat pathway. Optimal chaperone and signal peptide pairand optimal fermentation conditions for the translocation of GFP fusion protein via the Tatpathway will be determined. The results obtained in this study, we believe, will help us betterunderstand the controlling factors affecting the translocation of recombinant proteins via theTat pathway and facilitate the development of a feasible strategy for the expression andtranslocation of recombinant proteins that are incompatible with the Sec pathway.

近年來國際間對雙精胺酸轉位系統在大腸桿菌中之機制有極高之興趣。已有研究指出利用該系統搭配適當之大腸桿菌細胞,可選擇性地將具有完整雙硫鍵與輔因子之活性蛋白質輸送至細胞間質。由於將蛋白質輸送至細胞間質具有純化程序較簡單、可適度形成必須之雙硫鍵、較低之蛋白質水解速率等優點,因此是大量生產基因重組蛋白質一個可行當的方案。然而雙精胺酸轉位系統之作用機制目前並為完全被釐清,且其效率遠低於Sec 轉位系統,因此無論在學術上或在產業應用上都有必要針對輸送效率之提升建立一可行之策略,進而釐清雙精胺酸轉移系統之作用機制。本實驗室先前發現共表現TorD 可有效提升基因重組蛋白的輸送效率,並據以提出其作用機制,此項成果已獲得美、英、法、加國學者的支持並引用。我們最近更進一步發現共表現DmsD 亦可獲得相似的效果,並發現TorD 與DmsD 分別對DmsA signal peptide 與TorA signal peptide 具有交叉活性。本實驗室初步實驗進一步發現TorD 對其他signal peptide(如TorZ)亦有相同之效果;而DmsD 則無顯著效果,因此本計畫將針對大腸桿菌基因中29 個具有Tat signalpeptide 特性胺基酸序列之signal peptides,探討共表現TorD 與DmsD 對綠螢光蛋白在不同大腸桿菌寄主細胞轉位之影響,利用binding 實驗確認其作用機轉,進而建立最佳之E. coli Tat 表現輸送系統與最適化發酵條件。本計畫所獲得之研究結果除有助於釐清Tat 轉位路徑之控制機制,亦可能應用於無法以Sec 轉位路徑輸送之重組蛋白質於大腸桿菌之表現與輸送。
其他識別: NSC98-2221-E005-039
Appears in Collections:化學工程學系所

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