臺灣博碩士論文加值系統

本論文為研究樟芝子實體中之抗氧化酵素，榖氧還蛋白 (glutaredoxins, Grxs) 為具有硫氫基的氧化還原?，在細菌、酵母菌和哺乳動物中發現其主要的功能就是維持細胞內單硫鍵和雙硫鍵的平衡，近年來在真核和原核生物中發現 Grxs 之亞群，稱為單硫型榖氧還蛋白 (monothiol Grxs, monoGrxs)，但是其酵素特性尚未被完整的分析。本實驗利用聚合?連鎖反應，成功的自樟芝子實體中選殖出 monoGrx (TcmonoGrx) 之全長 cDNA 基因序列，基因全長 1037 bp，轉譯區 714 bp，可轉譯出 238 個胺基酸，分子量約為 27 kDa，此胺基酸序列與其他物種的 monoGrxs 之間具有高度的相似性，特別是如文獻所述之 Trx-Grx class 的 monoGrxs，具有 thioredoxin (Trx)-like domain (WAD/EPCK) 和 Grx domain (CGFS)。
以 pET-20b(＋) 為表現載體，Escherichia coli C41(DE3) 作為表現宿主，經由 IPTG 的誘發，即可表現大量的重組蛋白，而表現的重組蛋白TcmonoGrx 與 6 個 His-taq 所形成的融合蛋白，可利用含有鎳螯合物凝膠 (nickel chelating sepharose) 之親和性管柱來純化目標蛋白。
檢測酵素活性，TcmonoGrx 無法利用穀胱甘? (glutathione, GSH) 來還原 mixed disulfides，儘管將其 Grx domain 上的 CGFS168 突變為 CGFC168 也同樣無法檢測到活性；於 GSSG (oxidized GSH) reduction 的實驗中得知，TcmonoGrx 無須透過 thioredoxin reductase (TR) 就可以將GSSG 還原成 GSH，並利用 DNTB assay，進ㄧ步去證明 TcmonoGrx 會直接利用 NADPH 來還原 GSSG，且也推算出 TcmonoGrx 會利用一分子的 NADPH來還原ㄧ分子的 GSSG。另外，於檢測突變株活性之實驗中得知，TcmonoGrx 會利用 cysteine 進行 GSSG reduction，且 Grx domain 為其主要的活性區位。最後，從酵素動力學分析得到，TcmonoGrx 對 NADPH 的 Km 值為 0.041 mM，而對於 GSSG 的 Km 值為 0.064 mM。
觀察 TcmonoGrx 之基本特性，於 60 ℃ 之半衰期為 10.5 min；處在不同 pH 的環境下發現，其如同其他抗氧化酵素般，在鹼性環境下具有較高的活性，且能忍受的 pH 範圍很廣 (pH 4~10)；若以 0.2 M imidazole 或以上的濃度處理後，其活性皆剩下 60 %；加入 chymotrypsin 處理 40 min 後，活性仍有 70 %；而以 trypsin 處理 40 min 後，活性僅剩 50 %。

The purpose of this thesis is to study an antioxidant enzyme, glutaredoxins (Grxs) from Taiwanofungus camphoratus. Grxs are small thiol disulfide oxidoreductases. In bacterial, yeast and mammalian cells, Grxs appear to be involved in maintaining protein redox state. Recently, an emerging subgroup of Grxs with one cysteine residue in the putative active motif (monothiol Grxs, monoGrxs) has been identified in prokaryotes and eukaryotes but not well characterized. A cDNA encoding monoGrx from Taiwanofungus camphoratus fruit body (TcmonoGrx) was cloned by polymerase chain reaction (PCR). The DNA sequence has 1037 bp, which would encode a monoGrx of 238 amino acids with a molecular mass of 27 kDa. The deduced amino acid sequences is well-conserved among the reported monoGrxs, Trx-Grx class especially, including two domains : thioredoxin (Trx)-like domain (WAD/EPCK) and Grx domain (CGFS).
To further characterize the TcmonoGrx, the coding region was subcloned into an expression vector pET-20b(+) and transformed into Escherichia coli. The expression of the TcmonoGrx was purified by nickel chelating sepharose superflow column.
Analysis of TcmonoGrx activity, it was inactive as a glutathione (GSH)-disulfide oxidoreductase in a standard Grx assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and GSH reductase. An engineered CGFC168 active site of Grx domain mutant did not gain activity either. In GSSG (oxidized GSH) reduction and DTNB assay, TcmonoGrx was demonstrated to reduce GSSG to GSH by directly using NADPH, and the reduction corresponding to the consumption of one molecule of NADPH per molecule of GSSG. In analysis of mutants activity, TcmonoGrx was proved to use cysteine to reduce GSSG, and Grx domain was the major active site. The Michaelis constant (Km) values for NADPH and GSSG were 0.041 and 0.064 mM for the TcmonoGrx. The protein’s half-life of deactivation at 60 ℃ was 10.5 min. The enzyme activity was stable in a broad pH range from 4.0 to 10.0. The enzyme activity lost 40 % in the presence of 0.2 M or up to 0.8 M imidazole. Last, the enzyme showed 70 % and 50 % activity after 40 min of incubation at 37 ℃ with chymotrypsin and trypsin, respectively.

中文摘要………………………………………………………Ⅰ
英文摘要……………………………………………………... Ⅲ
縮寫表………………………………………………………… V
壹、前言………………………………………………………. 1
一、樟芝………………………………………………………………. 1
二、自由基……………………………………………………………. 6
三、穀氧還蛋白 (Grx)……………...……………………………….. 11
四、研究動機………………………………………………………… 16
貳、實驗材料………………………………………………… 25
一、原料……………………………………………………………… 25
二、載體……………………………………………………………… 25
三、宿主……………………………………………………………… 25
四、套組……………………………………………………………… 26
五、Maker…………………………………………………………….. 26
六、儀器設備……………………………………………………….... 26
七、實驗耗材……………………………………………………….... 27
八、實驗藥品……………………………………………………….... 28
九、引子...............…………………………………………………...... 30
參、實驗方法………………………………………………… 31
一、cDNA library之製備.…………………………………………..... 31
二、TcmonoGrx之基因選殖..……………………………….………. 33
三、TcmonoGrx之基因構築.…………………………………………38
四、TcmonoGrx之突變株製備....………………………………….... 42
五、TcmonoGrx之誘導表現與純化....……………………………… 42
六、TcmonoGrx之活性測定.………………………………………... 48
七、TcmonoGrx之酵素動力學研究..……………………………….. 52
八、TcmonoGrx之基本特性分析...….……………………………… 52
肆、結果………………………………………………………. 56
一、TcmonoGrx之基因選殖....……………………………………… 56
二、TcmonoGrx之基因構築.………………………………………. ..57
三、TcmonoGrx之誘導表現與純化..……………………………….. 58
四、TcmonoGrx之活性測定..…..…………………………………… 58
五、TcmonoGrx之酵素動力學研究....……………………………… 61
六、TcmonoGrx之基本特性分析.……………………………........... 62伍、討論……………………………………………………… 81
陸、參考文獻………………………………………………... 89
附錄………………………………………………………....... 99

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