利用融合蛋白技術製備動物生長激素及N端切醣酵素

Abstract

由於許多重組蛋白質水溶性差，造成在應用上之困難，故將其與能促進水溶性之融合蛋白一起表現，促使摺疊更加正確，進而提升水溶性，本實驗將麥芽糖結合蛋白（maltose binding protein）與不易表現且水溶性低的目標蛋白質利用基因重組技術融合為一，並藉由大腸桿菌大量表現出融合蛋白，期望進一步利用實驗室開發之自發斷裂胜肽技術以達大量製備重組蛋白的目標。 本研究使用了人工合成之牛生長激素（BST, bovine somatotropin）、豬生長激素（PST, porcine somatotropin）以及N-glycosidase之基因，利用基因重組技術將其分別插入pMAL-c2 Vector以構築所需之表現載體，再轉入BL21（DE3）大腸桿菌中表現，利用澱粉以及麥芽糖水溶液將其純化，所有融合蛋白均可達85%以上純度。 利用質譜儀確認其為正確之目標蛋白後，開始啟動自發斷裂反應可將目標蛋白與麥芽糖結合蛋白分離，結果顯示，經自發斷裂後之目標蛋白仍因水溶性低而沉澱，因此吾人決定將其保存環境調成pH 7 , 4℃，使融合蛋白可穩定保存而不會斷裂，並以融合蛋白進行各項活性測試。 我們以細胞生長情況、 IGF-I之濃度和細胞之醣質新生等實驗進行生長因子之活性測試，結果顯示，所得含生長因子之融合蛋白仍具備明顯的生理活性。N-glycosidase之活性則以多種醣蛋白，如β-xylosidase 和甜杏仁β-glucosidase為基質，並分別與市售之酵素比較，實驗結果顯示，含有訊息胜肽之自製的N-glycosidase具備活性，惟其活性比市售者弱了許多，其活性的提升仍有待後續力! 本研究成功展示藉由與麥芽糖結合蛋白形成融合蛋白的策略，可提高BST、PST和N-glycosidase之水溶性，同時可不需與麥芽糖結合蛋白分離仍具有生物活性。對於易形成包涵體之重組蛋白質而言，此研究提供一可行之解決方法。

Due to the poor solubility of some recombinant proteins, the applications of them have been limited. The strategy of forming recombinant fusion protein with maltose-binding protein to improve the solubility and biological activity of target protein is proposed in this study. Escherichia coli system integrated with the auto-cleavage peptide technique was employed for fusion protein expression. The auto-cleavage peptide technique, developed by our group, allows the fusion protein to break into apart at the desire position by controlling pH condition. Three synthetic genes including bovine somatotropin, porcine somatotropin, and N-Glycosidase are used as targets and inserted in pMAL-c2 vector for expression in BL21 (DE3). All three fusion proteins are found to be soluble. For all tested cases, after purifying by starch, the fusion protein can be obtained with 85% purity. The molecular weights were confirmed by ESI/MS. After auto-cleavage process to remove maltose-binding protein, the target proteins were found to be insoluble. For further study, the fusion proteins were kept at pH 7, 4℃to maintain their intact structures for biological assays. Three methods including cell growth status, the increasing IGF-I and observation of gluconeogenesis in cell were employed to evaluate the activity of growth factor. Results showed that growth factor (as fusion protein) revealed significant biological activity. By confirming the enzymatic activity of N-glycosidase, few glycoproteins, such as β-xylosidase expressed by Pichia pastoris and β-glucosidase from sweet almond, were used as substrate. By comparison the activity with that of commercial enzyme, we concluded the recombinant N-glycosidase with signal peptide is catalytically active though the activity is low. To improve the enzymatic activity of N-glycosidase will be the major issue for upcoming study of this subject. In summary, though the biological activities of the target proteins are not completely satisfied, this study does successfully demonstrate the maltose binding protein enable enhance protein solubility and folding, at least, for the cases of BST, PST and N-glycosidase. The study also provides a feasible solution for producing functional protein with hydrophobic nature.