目前水稻細胞生物反應器之蛋白質表達系統，乃應用脈-amylase 為啟動子加上分泌訊號蛋白片段接上所要表達的蛋白質基因，然後將基因送入水稻細胞中，並建立轉殖懸浮培養細胞系，經過大量培養的過程，最後從培養液中得到所要表達的蛋白質。雖然，目前的脈-amylase 分泌訊號蛋白可以把重組蛋白質帶到細胞外，但是有時分泌到培養液的重組蛋白質產量不高。分析比較細胞內與細胞外重組蛋白質的量，發現30%~50%的重組蛋白質並未分泌至細胞外。因此，為了提高重組蛋白質分泌至培養液的比例，並應用於水稻細胞生物反應器，我們將進行一連串的研究與開發，期望可以將85%以上甚至 100%的重組蛋白質都分泌到培養液中，因為此計畫先前已經初步篩選到較高效率的分泌訊號蛋白(Os33KDa)，為了要確定不同分泌訊號蛋白分泌mGM-CSF 的效率，我們也將持續進行mGM-CSF 轉殖株的分析。因此，此計畫將分為3 個階段來進行: 1.分析分泌訊號蛋白分泌mGM-CSF 的效率。監測與分析mGM-CSF 在細胞內與細胞外的表現量，如此我們可以篩選到最有效率的分mGM-CSF 泌訊號蛋白2.篩選高效率人工改造的分泌訊號蛋白。建立可分析分泌效率的短暫表現系統，以縮短分析的時間與增加篩選分泌訊號蛋白的數量。然後再針對效率最佳的訊號蛋白作人為改造，使得此訊號蛋白的分泌效率能夠進一步的提升，達到可以將85%以上甚至100%的重組蛋白質都分泌到培養液中。3. 整合三個子計畫的成果，應用於mGM-CSF 生產。整合另外兩個子計畫的成果，我們將把最佳與次佳的分泌訊號蛋白接到mGM-CSF 的N 端，然後由接上第一子計畫所得到的最高活性與最高誘導性的啟動子，再將這些基因轉殖進入由第三子計畫所得到的低蛋白質分解酵素的水稻細胞株，進一步建立這些轉殖細胞系，監測這些轉殖細胞mGM-CSF 的表現量，由此可以得到最大表現量的轉殖細胞系。將所得到的轉殖細胞系建立實驗室規模量產生化反應器，建立最佳的生產模式，達到實驗室量產規模。 As a bio-factory, rice suspension cells have provided an attractive strategy to produce valuable recombinant proteins. Basically, foreign proteins are expressed and secreted into culture medium, which is benefit for down stream protein purification, from a transformed rice suspension cell line carrying an 脈-amylase promoter fused with its signal peptide and a target foreign gene. However, the signal peptide of 脈-amylase is not the best choice for secretion of recombinant protein. 30~50% recombinant proteins were still left inside of rice cells when 脈-amylase signal peptide was applied. In order to improve the secretion efficiency of recombinant proteins in rice expression system, we are going to screen signal peptides for better secretion efficiency than 脈-amylase signal peptide. We expect selected signal peptides are able to secret more, at least 85% of target proteins, recombinant proteins into culture medium and also suitable used in rice suspension cell bioreactor. Previously, we have identified the Os33kDa signal peptide secretory efficiency was higher than OsCIN and 脈Amy3.It is important to conform that Os33kDa signal peptide also have higher ability to secrete mGM-CSF. The proposal provided here contains three parts. First, we are going to analysis recombinant proteins in mGM-CSF transformates to identify secretion efficiency of signal peptides. Second, we are going to made artificial signal peptides with high efficiency. The Os33kDa signal peptide amino acids will be changed by serial mutagenesis and fused with reporter gene (GFP) under a constitute promoter. The secretion efficiency of each artificial signal peptide will be compared using transient expression system. Further, the highest efficiency signal peptides is going to secret mGM-CSF recombinant proteins in rice suspension cells. Third, combining conclusions from other two related projects, a high transcriptional activity promoter with high efficiency signal peptide under low proteinase activity rice host cell will be developed for production of mGM-CSF recombinant proteins. Finally, a lab-scale mGM-CSF producing bioreactor will be built up. 研究期間：9601 ~ 9612
