Cloning and expression of the glucose-6-phosphatase catalytic subunit 1 and enzymatic characterization of avtive site
張容甄、簡宏堅
E-mail: [email protected]
ABSTRACT
Human glycogen storage disease type 1 (namely GSD-1a),also known as von Gierke disease, is a group of autosomal recessive disorders of glucose-6-phosphatase (G6PC) genes with an overall incidence of approximately 1 in 100,000. Glucose-6-phosphatase (G6PC) is a key enzyme in glucose homeostasis that catalyzes the hydrolysis of glucose-6-phosphate to glucose and phosphate in the terminal step of gluconeogenesis and glycogenolysis. G6PC was recently divided into three subunits. Mutations in the G6PC gene, located on chromosome 17q21, result in disorder of the subunit 1 of glucose-6-phosphatase. Major clinical syndromes of G6PC1 mutants are hypoglycemia, hypercholesterolemia, hypertriglyceridemia,hyperuricemia, lactic acidemia, hepatomegaly,
nephromegaly, fat liver and hepatoma (G6PC1). Because there is no medicine that can treat GSD-1a disease, only relying on the future supply of nutrients to improve the relief of symptoms. If the depth of G6PC1 to be able to study if the activity location, selection of high activity G6PC1 enzymes, overexpression and purification, to produce highly active enzyme, as a treatment glycogen storage disease type I protein drug development. The molecular weight of 58 kDa, because the pQE30 expression vector that contains six Histidine (His-tag), it can be purified easy in the future, then the method using NAD to analyze the enzyme activity. The wild-type enzyme in the pH value 6.5 of phosphate buffer (100 mM) and 37 ℃, and 5 mM G6P catalyzed reaction for 30 minutes, with optimum response. We observed L31 (I198F) and L21 (V318A) mutants have lower activity than wild- type kcat / Km in 8.2 fold and 10.3 fold, respectively .The L21(V318A),We also found that H4 (S196N L305P) mutant has higher activity than wild type in 8 fold, We observed H19 (S196G) and H2 (N203S) mutant has higher activity than wild type in 1.2 fold and 4 fold, respectively. We observed modeling of E.coil glucose-1-phosphatase active center is similar to G6PC1. According to modeling of structure from E.coil glucose-1-phosphatase active center, corresponding L305P was found very close to the phosphatase hydrolysis site. Other mutant I198F, V318A, N203S, S196G and S196N were on the peripheral of entrance in the active center that might change the original amino acid and influenced substrate binding for G6PC1 activity. In this study, data suggest L305P are catalytically important active site of G6PC1. We are going to add enzyme of G6PC1 into yogurt making a medicine that can be a treatment of glycogen storage disease la in the future.
Keywords : glycogen storage disease la、von Gierke disease、Glucose-6-phosphatase catalytic subunit 1、Specific activity、active site、active center
Table of Contents
目錄 封面內頁 簽名頁 授權書...iii 中文摘 要...iv 英文摘
要...vi 誌 謝...viii 目 錄...ix 圖目 錄...xii 表目 錄...xiv 1. 前
言...1 1.1 肝醣儲積症...1 1.2 葡萄糖六磷酸酵素次單元一的功能...2 1.3 利用酵素的方式生產天然產物的優點及缺點...3 2. 研究 動機...4 3. 材料與方
法...5 3.1 材料...5 3.1.1 菌種 及質體...5 3.1.2 藥品...6 3.1.3 酵
素...6 3.1.4 培養液...6 3.1.4.1 LB 培養 基...7 3.1.5 其他緩衝液及試劑...8 3.1.6 引子設
計...12 3.2 實驗方法...13 3.2.1 製備G6PC1 質體DNA...13 3.2.2 聚合?連鎖反應...15 3.2.2.1聚合?連鎖反 應...15 3.2.2.2 隨機突變聚合?連鎖反應... 16 3.2.3 DNA洋菜膠
體...17 3.2.4 DNA片段的回收及純化...19 3.2.5 限制酵素剪切
…...20 3.2.6 DNA黏接作用...20 3.2.7 E. coli勝任細胞的製 備...20 3.2.8 E. coli的轉形作用...21 3.2.9 E. coli質體DNA的抽 取...22 3.2.10 快速質體抽取套組...23 3.2.11 隨機突變葡萄糖六磷酸?次 單元酵素之小量菌體 活性篩選...24 3.2.12 DNA 定
序...24 3.2.13葡萄糖六磷酸?次單元蛋白質表現及蛋白回收...25 3.2.14 聚丙烯醯胺凝 膠電泳分析...26 3.2.15 G6PC1酵素活性分析測定...27 3.2.15.1 G6PC1最適反應 時間的測定...27 3.2.15.2 G6PC1酵素最適pH值的測定...27 3.2.15.3 G6PC1酵素最適溫度的測 定...28 3.2.15.4 G6PC1基質抑制作用測定...28 3.2.15.5 G6PC1產物抑制作用測
定...29 3.2.15.6 標準曲線的製作...29 4. 結
果...31 4.1 製備G6PC1質體DNA與基因分析…...31 4.2 葡萄糖六磷酸?次單元酵素的蛋白質表現與分析...31 4.3 野生型葡萄糖六磷酸次單元一酵素活性分析...32 4.3.1 葡萄 糖六磷酸?次單元一酵素最適pH值...32 4.3.2 葡萄糖六磷酸?次單元一酵素最適溫度...32 4.3.3 葡萄糖六磷酸?次單 元一酵素最佳反應時間...32 4.3.4 G6PC1最適受質濃度及抑制作用測定...33 4.3.5 G6PC1產物抑制作用測
定...33 4.4 隨機定點突變葡萄糖六磷酸?次單元一酵素的活分析
...33 4.4.1 Error-prone PCR隨機定點突變 ...33 4.4.2 葡萄糖六 磷酸?次單元一酵素菌株的蛋白質大量 表現及回收...34 4.4.3 Wild type以及突變葡萄糖六 磷酸?的活性分析...35 5. 結論...37 5.1 G6PC1的酵素活性特
性...37 5.2 G6PC1基因之應用...37 5.3 模擬G6PC1活性 中心結構之探討的關係...38 參考文獻...69 圖目錄 圖1.G6PC1酵素之活性測定實驗設計...39 圖2. G6PC1基因並轉殖於pQE30表現載體
中...40 圖3. G6PC1基因plasmid DNA的膠體電泳...41 圖4. G6PC1酵素聚合?連鎖反 應膠體電泳………...42 圖5. G6PC1酵素基因核酸序列比對...43 圖6. 聚合?連鎖反 應後DNA回收之膠體電泳...44 圖7. 野生型G6PC1胺基酸序列...45 圖8. 軟 體分析G6PC1酵素之胺基酸百分比...46 圖9. 野生型G6PC1基因蛋白回
收...47 圖10.G6PC1酵素最適pH緩衝溶液...48 圖11.G6PC1酵素最 適溫度...49 圖12.G6PC1酵素最適反應時間...50 圖13.
G6PC1酵素活性對受質的抑制作用...51 圖14. G6PC1酵素產物抑制作
用...52 圖15. 突變G6PC1基因藉由Error-prone PCR膠體電泳...53 圖16. PCR膠體電 泳回收...54 圖17. 篩選隨機突變G6PC1...55 圖18.
突變G6PC1菌株轉譯成胺基酸之定序結果...56 圖19. I198F和V318A兩隻低活性和S196N、L305P和N203S三 隻高 活性的G6PC1突變菌回收...58 圖19-A. 利用SDS-PAGE測定突變I198F G6PC1菌株蛋白 質回收..58 圖19-B. 利用SDS-PAGE測定突變V318A G6PC1菌株蛋白質回收
...59 圖19-C. 利用SDS-PAGE測定突變S196N L305P G6PC1菌株蛋白 質回收...60 圖19-D. 利用SDS-PAGE測定突變S196N G6PC1菌株蛋白質回收 ...61 圖19-E. 利用SDS-PAGE測定突變N203S G6PC1菌株蛋白 質回收 ...62 圖20. 利用Bio-Rad assay 製作出BSA標準曲線定 量蛋白質...63 圖21. 野生型G6PC1與突變I198F和V318A、以及S196N、L305P 及N203S G6PC1菌株之酵素動力學分 析...64 圖 22.藉由Glucose Dehydrogenase-Coupled Reaction方法製作出此Glucose標準曲
線...66 圖23. E. coli glucose-1-phosphatase 結構...67 圖24. 模 擬G6PC1活性中心電腦模擬結構...68 表目錄 表1. 菌種與質
體...5 表2. LB液態培養基配方...7 表3.
LA固態培養基配方...7 表4. 引子設
計...13 表5. SOC液態培養基配方...22 表6. 比較野生型與突變G6PC1基因酵素動力學分析...36
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