Production of functional plant farnesylated proteins in Escherichia coli

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Title:	Production of functional plant farnesylated proteins in Escherichia coli
Authors:	左瑞塔;Zohra, Rida
Contributors:	生命科學系
Keywords:	法尼基化;Farnesylation
Date:	2024-06-18
Issue Date:	2024-10-09 15:26:25 (UTC+8)
Publisher:	國立中央大學
Abstract:	蛋白質法尼脂化是將一個含有15個碳的不飽和法尼脂肪基團，以共價鍵的方式添加到C-端以CaaX序列結束的蛋白質上。這是轉譯後蛋白質修飾的一種形式，由蛋白質法尼脂轉移酶（Protein farnesyltransferase, PFT）催化進行。完整的PFT由α和β兩個次單元所組成。蛋白質法尼脂化在植物生長過程中扮演非常重要的角色，它在調節植物發育、對抗環境逆境，抵禦病原體感染方面發揮重要作用。傳統上，用於檢驗某候選蛋白質是否被法尼脂化的方法，需要先製備該候選蛋白質的專一性抗體，並使用到同位素標記金合歡焦磷酸 (farnesol, 為蛋白質法尼脂化反應中法尼脂焦磷酸 farnesyl pyrophosphate的前驅物)，無疑是一個費時費力且存具有安全風險的程序。由於蛋白質法尼脂化不發生在原核細胞中，本研究目的即是在大腸桿菌中，建立一個可以快速鑑別植物候選蛋白質被法尼脂化與否的系統。該系統在大腸桿菌中同時殖入一帶有ColE1複製起始點(Ori)，攜帶阿拉伯芥PFT的α和β次單元基因的質體 (plasmid)，及一帶有RSF1031複製起始點，攜帶被6xHis序列標記之待檢驗蛋白質基因的質體。本研究選用一已知為阿拉伯芥PFT受質的蛋白質AtJ3，作為待檢驗基因。結果顯示，三個阿拉伯芥基因在大腸桿菌細胞內可同時表達，所製造出之At3J經過簡單的親和性純化後，藉由SDS-PAGE測其泳動速率，即可辨識出AtJ3轉譯後有被修飾，且質譜分析確認該修飾為C端接上法尼脂分子。AtJ3是熱休克蛋白40家族的成員，與HSP70相互作用，以保護植物蛋白質免於高溫造成的變性傷害。基於螢光素酶 (luciferase)活性分析實驗，本研究也證明從大腸桿菌中分離出的法尼脂化AtJ3，保有其分子伴侶功能。有趣的是，在大腸桿菌製造出的法尼脂化AtJ3，也能與大腸桿菌HSP70相互作用，並提升大腸桿菌的耐熱能力。此外，使用相同的策略共同在大腸桿菌細胞內表達水稻PFTα和β次單元及一個潛在的水稻PFT受質OsDjA4，結果也顯示OsDjA4有被法尼脂化。綜上述，本研究成功開發了一種利用大腸桿菌鑑識蛋白質是否有被法尼脂化修飾的系統，該系統適用於單子與雙子葉植物，且所產生之法尼脂化蛋白質，保有其在真核細胞內的原有功能，可應用於研究法尼脂化蛋白質分子與生化功能。;Farnesylation of proteins involves the covalent attachment of a 15-carbon unsaturated farnesyl lipid group to a protein ending with a CaaX sequence at the C-terminus. This post-translational modification is catalyzed by the protein farnesyltransferase (PFT), which comprises α and β subunits. Protein farnesylation is pivotal in plant growth, regulating plant development, combating environmental stresses, and defending against pathogen infections. Traditionally, assessing whether a candidate protein is farnesylated involves laborious and potentially hazardous processes, such as preparing specific antibodies for the candidate protein and utilizing isotopically labeled farnesol, a precursor of farnesyl pyrophosphate in the farnesylation reaction. Given that protein farnesylation does not occur in prokaryotic cells, this study aims to establish a system in E. coli for rapidly identifying whether plant candidate proteins are farnesylated. This system involves introducing into E. coli a plasmid carrying the ColE1 replication origin, the α and β subunit genes of Arabidopsis PFT, and a plasmid carrying the candidate protein gene labeled with a 6xHis sequence, along with the RSF1031 replication origin. This study selected a known substrate of Arabidopsis PFT, AtJ3, as the candidate protein. The results demonstrate that all three Arabidopsis genes can be expressed simultaneously in E. coli cells. The produced AtJ3, after simple affinity purification, can be identified as post-translationally modified by its mobility on SDS-PAGE, with mass spectrometry confirming the modification as farnesylation at the C-terminus. AtJ3 belongs to the heat shock protein 40 family, interacts with HSP70, and protects plant proteins from denaturation damage caused by high temperatures. Based on luciferase activity analysis experiments, this study also demonstrates that farnesylated AtJ3 isolated from E. coli retains its molecular chaperone function. Interestingly, farnesylated AtJ3 produced in E. coli can also interact with E. coli HSP70 and enhance the thermotolerance of E. coli. Furthermore, using the same strategy to co-express rice PFTα and β subunits along with a potential rice PFT substrate, OsDjA4, in E. coli cells also showed OsDjA4 to be farnesylated. In summary, this study successfully developed a system using E. coli to identify protein farnesylation modifications, applicable to both monocot and dicot plants. The farnesylated proteins produced in E. coli retain their original functions in eukaryotic cells and can be utilized to study the molecular and biochemical functions of farnesylated proteins.
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