利用Tsukuba System於菸草中實現瞬時蛋白質表達

Abstract

本研究以本實驗室於日本開發之瞬時蛋白質表現系統「Tsukuba System」為核心進行。透過雙聯學位計畫，本人分別於日本與臺灣兩地進行研究。在日本，嘗試生產並進行流感抗原特異性 MHC 之四聚體化；在臺灣，則嘗試生產與植物免疫相關之蛋白質。目標基因分別插入源自 Tsukuba System 骨幹載體 pTKB3 之內質網（ER）滯留訊號載體（pTKB3-SSExt-HKD）或質外體運輸訊號載體（pTKB3-SSExt-CSPH）中。上述載體轉形至 Agrobacterium tumefaciens GV3101，並透過農桿菌浸潤法（agroinfiltration）於 Nicotiana benthamiana 中進行瞬時表現。隨後將葉片組織均質化並過濾以萃取目標蛋白質，並以 SDS-PAGE 分析確認所得蛋白之存在。 日本之研究內容詳述於「Chapter 2」，臺灣之研究內容則說明於「Chapter 3」。

【Chapter 2】 重組蛋白廣泛應用於功能分析與診斷試劑開發等領域。一般而言，重組蛋白多利用 Escherichia coli 等異源表現系統進行生產。然而，部分蛋白質，尤其是結構複雜之蛋白，在 E. coli 系統中表現困難；此外，植物來源蛋白亦常面臨表現效率不佳之問題。為克服上述限制，本實驗室建立了一套植物表現系統—「Tsukuba System」。此系統結合源自雙生病毒（geminivirus）之滾環複製機制（rolling circle replication）與雙終止子（double terminator）設計。相較於既有之 magnICON 植物瞬時表現系統，Tsukuba System 可達成更快速且高產量之蛋白質瞬時表現。

本研究旨在利用 Tsukuba System 表現並分析 MHC class II 蛋白與植物免疫相關蛋白之功能，兩者皆屬於難以在 E. coli中大量生產之蛋白質。

MHC 蛋白為評估免疫反應之關鍵分子，並具疫苗開發潛力。由於抗原具高度多樣性，需製備對應不同抗原之 MHC 蛋白。雖然 MHC class I 蛋白於 E. coli 表現系統中已有成熟技術，然而 MHC class II 蛋白之製備仍具挑戰性，目前僅能生產有限類型。此外，MHC 分子於體內可與 T 細胞受體結合，但此結合易迅速解離。若應用於診斷，則需更穩定之結合形式。形成 MHC 四聚體為提升結合穩定性之一種策略，可藉由增加結合位點數目以降低快速解離之機率。 MHC 單體由 α 鏈與 β 鏈組成。於 N. benthamiana 中共表現 α 與 β 鏈後，透過與 α 鏈融合之 His tag 進行純化，確認 β 鏈可共同被純化，顯示 MHC 單體複合體成功形成。隨後將所得 MHC 單體進行生物素化（biotinylation），並與 avidin 結合形成四聚體。四聚體化後之 SDS-PAGE 分析顯示約 100–200 kDa 之條帶，證實多聚體之形成。此階段之蛋白產量為每 100 g 新鮮葉片約 50 ng。

Tsukuba System 成功於 N. benthamiana 中高效率生產 MHC 單體，然而四聚體化仍具挑戰性。雖觀察到多聚體條帶，但未明確檢測到預期之四聚體條帶，顯示組裝可能不完全或不正確。此現象可能源於生物素化 MHC 單體僅與 streptavidin 之兩至三個結合位點結合，或游離生物素佔據 streptavidin 結合位點，導致無法有效結合生物素化 MHC 單體。 為提升 MHC 四聚體形成效率，可於與 streptavidin 結合前，先依分子量分離生物素化單體，以利更有效率地分離與純化四聚體。

【Chapter 3】 植物免疫蛋白在植物抵禦病原菌過程中扮演關鍵角色，其功能鑑定對於理解植物防禦機制至關重要。然而，部分植物來源蛋白難以於 E. coli 中有效表現。本研究聚焦於三種番茄來源之免疫相關蛋白：SlPNGase（peptide:N-glycanase）、SlWfi1，以及功能尚未明確之 12g520。上述基因先前已被鑑定為參與番茄病原菌反應之相關基因。 SlWfi1 為定位於細胞膜之 NADPH oxidase，於病原入侵時可誘導活性氧（ROS）生成及細胞死亡反應，在防禦訊號傳遞中扮演核心角色。SlPNGase 屬於 PNGase 酵素家族，可去除醣蛋白之 N-連結型醣鏈，推測在病原反應過程中參與蛋白質品質管制。12g520 為一 LRR receptor-like 基因，係透過 Bwr12 抗性數量性狀基因座（QTL）區域之比較基因體分析所鑑定，推測參與病原相關分子模式（PAMP）辨識及啟動 PAMP-triggered immunity（PTI）反應。上述基因係本研究室於國立臺灣大學進行研究時，自萃取 RNA 合成 cDNA 後所擴增取得。本研究之目的在於利用 Tsukuba System 表現上述蛋白，並進行功能分析。 於植物免疫蛋白之製備方面，目標基因被克隆至 pTKB3-SSExt-HKD 與 pTKB3-SSExt-CSPH 載體，並轉形至 E. coli。雖成功萃取質體 DNA，然而其濃度未超過 10.0 ng/μl，不足以進行後續農桿菌轉形與 agroinfiltration 實驗。

在植物免疫相關蛋白表現方面，質體 DNA 產量偏低導致轉形效率受限。儘管參考臺灣之實驗流程進行多項條件優化，包括改變培養條件與增加通氣量，仍未顯著改善結果。E. coli 菌株差異及培養條件不同可能為影響因素。 此外，由於植物免疫相關蛋白之目標基因序列較大，需反覆確認載體建構之正確性。先前於臺灣之研究指出，除 pTKB3 之外之其他載體無法於 N. benthamiana 中成功表現上述蛋白。因此，未來將持續以 pTKB3 載體系統為核心，建立穩定且可靠之蛋白表現條件。

This research focuses on the transient protein expression system, the “Tsukuba System,” developed in our laboratory in Japan. Through a double degree program, I conducted research in both Japan and Taiwan. In Japan, I produced and tetramerized influenza antigen-specific MHC, while in Taiwan, I produced plant immunity-related proteins. Target genes were inserted into either the ER retention signal vector (pTKB3-SSExt-HKD) or the apoplast transport signal vector (pTKB3-SSExt-CSPH), both derived from the pTKB3 backbone of the Tsukuba System. The vectors were introduced into Agrobacterium tumefaciens GV3101 and transiently expressed in Nicotiana benthamiana via agroinfiltration. Leaf tissue was homogenized and filtered to extract proteins, which were confirmed by SDS-PAGE. Research conducted in Japan is described in Chapter 2, and research in Taiwan in Chapter 3.

【Chapter 2】 Recombinant proteins are widely used in applications. Typically, heterologous protein expression systems like Escherichia coli are employed for recombinant protein production. However, certain proteins, particularly complex ones, are difficult to express in systems such as E. coli. Additionally, plant-derived proteins can also pose challenges in expression. To overcome these limitations, our laboratory has developed a plant-based protein expression system called as “Tsukuba System”. MHC proteins are essential for evaluating immune responses and have potential applications in vaccine development. Because of antigen diversity, various antigen-specific MHC proteins are required. Although E. coli-based expression systems for MHC class I are well established, production of MHC class II remains limited. In addition, MHC molecules dissociate rapidly from T cell receptors in vitro, which limits diagnostic use. To improve stability, MHC tetramers can be formed to increase binding sites and reduce dissociation. MHC monomers consist of α and β chains. Co-expression of both chains in N. benthamiana followed by His tag purification of the α chain confirmed co-purification of the β chain, indicating successful complex formation. The monomers were biotinylated and conjugated to avidin to generate tetramers. SDS-PAGE showed bands at approximately 100–200 kDa, suggesting multimer formation. The protein yield was 50 ng per 100 g of fresh leaf tissue. The Tsukuba System enabled efficient production of MHC monomers in N. benthamiana, but tetramerization remains challenging. Although multimer bands were observed, a clear tetramer band was not detected, suggesting incomplete assembly. Possible causes include partial binding of biotinylated monomers to streptavidin or occupation of streptavidin binding sites by free biotin. To improve tetramer formation, separating biotinylated monomers by molecular weight before streptavidin conjugation may enhance tetramer isolation.

【Chapter 3】 P Plant immune proteins play a vital role in protecting plants from pathogens, making their functional characterization crucial. However, some plant-derived proteins are difficult to express in E. coli. In this study, I focus on three tomato-derived immune proteins, SlPNGase (peptide:N-glycanase), SlWfi1, and the functionally uncharacterized 12g520, previously identified as pathogen-responsive genes. SlWfi1 is a membrane-localized NADPH oxidase that induces reactive oxygen species (ROS) production and cell death upon pathogen invasion. SlPNGase belongs to the PNGase family, which removes N-linked glycans from glycoproteins and contributes to protein quality control during pathogen response. 12g520 is an LRR receptor-like gene identified in the Bwr12 resistance QTL region and is thought to be involved in pathogen-associated molecular pattern (PAMP) recognition and activation of PAMP-triggered immunity (PTI). The genes were previously amplified from tomato cDNA synthesized from RNA in research conducted at National Taiwan University. The aim of this study is to perform functional analysis by expressing these proteins using the Tsukuba System. The target genes were cloned into pTKB3-SSExt-HKD and pTKB3-SSExt-CSPH and transformed into E. coli. Although plasmid extraction was successful, DNA concentration did not exceed 10.0 ng/μl, which was insufficient for Agrobacterium-mediated agroinfiltration. Low plasmid yield hindered successful transformation. Despite modified culture conditions and increased aeration based on Taiwanese protocols, no significant improvement was observed, possibly due to differences in E. coli strains and growth conditions. Because the target genes are large, repeated verification of vector construction is required. Previous research in Taiwan showed that vectors other than pTKB3 failed to express these proteins in Nicotiana benthamiana. Therefore, future efforts will focus on achieving reliable expression using the pTKB3 vector system.