6-1 15N- Aβ40之生產與純化
為進行1H-15N 之 HSQC 實驗，必須要能夠先生產並純化15N- Aβ40，本此純化 流程中可發現，以1L 的 M9 培養液可成功獲得~0.6mg 之融合蛋白，若能達到 100%
之TEV 蛋白脢切割率則可獲得~0.4mg 之 Aβ40，然而此次純化中遭遇到兩個問題，
分別是TEV 蛋白脢之切割率極低，以及 HPLC 無法將融合蛋白與 Aβ40分離。
由同樣以TEV 蛋白脢切割並純化 Aβ 之先前文獻83中發現，4℃隔夜應為充分
之反應時間及反應溫度，本次純化切割率過低的問題應是由於所添加的TEV 蛋白
脢不足量，無法與獲得之融合蛋白完全反應，因此應先進行蛋白脢與融合蛋白之 混合比例測試，以確認能夠充分反應之TEV 蛋白脢與融合蛋白比例。
而HPLC 無法將融合蛋白與 Aβ40分離之問題，可藉由再次以鎳親和管柱純化 以將Aβ40與未切割之融合蛋白與切割下之His-tag 部分分離。再次以 HPLC 純化應 可預期得到高純度之Aβ40。
此外為提升純化之產率，在各個步驟皆應避免產物的損失，如透析置換緩衝 溶液的步驟，應避免將融合蛋白溶液之濃度提升太多，以避免形成沉澱而造成損
失。在進行TEV 蛋白脢之切割時，可使用攪拌子以確認蛋白脢與融合蛋白充分混
合，提升剪切之成功率。之後在進行其他突變型Aβ40之純化時也應重新測試如表 現等級、蛋白脢添加比例等純化條件，以獲得最大可能之產率。
6-2 以光譜法檢測原生型及突變型 β-類澱粉蛋白與金屬之結合關係
比較不同公式後發現，由Total binding equation 所 fitting 之結果最能充分描述 β-類澱粉蛋白與金屬離子的結合關係，相較於先前文獻84中之原生型β-類澱粉蛋
6-3 以恆溫滴定量熱儀檢測原生型及突變型 β-類澱粉蛋白與金屬之結合關係
此處獲得之金屬離子與原生型及突變型β-類澱粉蛋白之計量數(stoichiometric number)皆在 0~1 之間。然而所得結果並無法確定金屬與 β-類澱粉蛋白之鍵結關係
附錄
附錄A 利用 Quick Change Primer Design Program 設計引子
1. 首先連結到https://www.genomics.agilent.com/ ，註冊並登入網站。
2. 選取 Applications→Mutagenesis，並點選 Quick Change Primer Design Program。
3. 選取所使用的 Site-directed mutagenesis kid 名稱，此處以 QuickChangeII 為例。
4. 將所使用之 DNA template 序列以 FASTA 格式貼上在表格處。
5. 點選 Upload 上傳 DNA 序列。
6. 上傳完成後頁面會自動更新並顯示各個位置之 DNA 序列。
7. 選擇要進行 Change、Delete、或是 insert DNA 序列，此處我們要進行的是突 變因此選擇Change。
8. 勾選要進行突變之 DNA 位置，並選取所要變更的 DNA 序列，例如要將線嘌 呤(A)突變成胞嘧啶 (C)，則勾選所要突變位置後在 site 1 的下拉式選單中選取 C。
9. 按下 Design Primer，程式就會自行設計所需的正、反向引子。
10. 此處以 D7A 為例，結果如下頁所示。
表A-1、Quick Change Primer Design Program 結果
Primer sequences:
Primer Name Primer Sequence (5' to 3') D13A 5'-gatgcagaattccgacatgcctcaggatatgaagttcat-3' D13A_antisense5'-atgaacttcatatcctgaggcatgtcggaattctgcatc-3'
Oligonucleotide information:
Primer Name Length (nt.) Tm Duplex Energy at 68°C
Energy Cost of Mismatches
D13A 39 78.45°C -44.91 kcal/mole 3.2%
D13A_antisense 39 78.45°C -46.45 kcal/mole 1.2%
Primer-template duplexes:
Primer Name Primer-Template Duplex
D13A
附錄B 以自動滴定儀進行去摺疊與再折疊實驗
為達到快速、精準的目的，嘗試以自動滴定儀Microlab500搭配 螢光光譜儀 Fluormax-3 進行自動化蛋白質摺疊與去摺疊實驗。首先選取已被廣泛用來進行蛋 白質去折疊實驗之核糖核酸酶A(RNaseA, Roche, 70297724)進行測試，並選用 130mM Tris-HCl, pH7 做為緩衝溶液，以及胍鹽酸(GdnHCl)做為去活性劑。先將 RNaseA 以 1mg/ml 的濃度溶解於緩衝溶液。再分別將其以 0.05mg/ml 濃度加入緩 衝溶液及8M 胍鹽酸至以配製原態(Native stage)及去折疊態(Unfolded stage)之溶 液。
去摺疊實驗操作：
先將0.05mg/ml RNaseA 溶液 1.5ml 置於 1cm 光徑 4ml quartz cuvette 或取 0.6ml 放入0.7ml quartz cuvette，接著放入 Fluormax-3 (Horiba Jobin Yvon)。將含有 0.05mg/ml RNaseA 之 8M 胍鹽酸填充於自動滴定儀之管柱 A。再將連結管柱 A、B (Bigelow, C.C. (1982) J. Biol. Chem. 257, 12935-12938)，可看到圖 B-4 中去折疊與再 折疊曲線幾乎完全重合，且與圖B-6 相近，證實此設置是可行之做法，唯所需蛋 白質樣品耗費過多，因此進一步使用0.7ml cuvette 進行實驗，以降低樣品需求量。
圖B-5 為使用 0.7ml cuvette 進行之自動滴定結果，與圖 B-4 相較，此設定下
隨機誤差對結果產生之影響較大。但去折疊與再折疊曲線仍幾近重合，且與圖B-6
相近，證實此自動滴定裝置之設置為可行之實驗方法。
圖B-1、自動滴定裝置設定示意圖
圖B-2、去折疊實驗光譜
圖B-3、再折疊實驗光譜
圖B-4、以 GdnHCl 進行之 RNaseA 滴定曲線(4ml cuvette)
圖B-5、以 GdnHCl 進行之 RNaseA 滴定曲線(0.7ml cuvette)
圖B-6、文獻之滴定曲線
附錄C TEV-Aβ40-pET14b 質體之序列
TEV-AB40-pET14b
4329 bp
Abeta40
HisTag*6
TEV cleavage site
Hin dIII (4297)
XhoI (4153)
圖C-1、TEV-Aβ40-pET14b 質體之序列
附錄D 自身螢光滴定曲線與 Total binding fitting 曲線
圖D-2、以 total binding equation fitting 之鋅離子與 Aβ40 WT 滴定曲線(上)及
圖D-3、以 total binding equation fitting 之銅離子與 Aβ40 D7A 滴定曲線(上)及
圖D-4、以 total binding equation fitting 之鋅離子與 Aβ40 D7A 滴定曲線(上)及
圖D-5、以 total binding equation fitting 之銅離子與 Aβ40 D7H 滴定曲線(上)及
圖D-6、以 total binding equation fitting 之鋅離子與 Aβ40 D7H 滴定曲線(上)及
圖D-7、以 total binding equation fitting 之銅離子與 Aβ40 D7N 滴定曲線(上)及
圖D-8、以 total binding equation fitting 之鋅離子與 Aβ40 D7N 滴定曲線(上)及
圖D-9、以 total binding equation fitting 之銅離子與 Aβ40 H6A 滴定曲線(上)及
圖D-10、以 total binding equation fitting 之鋅離子與 Aβ40 H6A 滴定曲線(上)及
圖D-11、以 total binding equation fitting 之銅離子與 Aβ40 H6R 滴定曲線(上)及
圖D-12、以 total binding equation fitting 之鋅離子與 Aβ40 H6R 滴定曲線(上)及
附錄E 縮寫表
全名 縮寫
Amyloid precursor protein APP 1,4 Dithiothreitol DTT 4,4'-bis(1-anilino-8-naphthalene sulfonate) Bis-ANS Ammonium persulfate APS
Amyloid beta Aβ
Bicinchoninic acid assay BCA blood brain barrier BBB Brovine serum albumin BSA Circular Dichroism CD
Electrochemiluminesence ECL electron paramagnetic resonance EPR
familial Alzheimer’s disease FAD Fast protein liquid chromatography FPLC Heteronuclear Single Quantum Correlation HSQC High-performance liquid chromatography HPLC Isopropylthio-β-galactoside IPTG Isothermal Titration Calorimetry ITC Luria-Bertani broth LB broth mass spectrometry MS Nuclear Magnetic Resonance NMR photo-induced cross-linking of unmodified
proteins
PICUP
reactive oxygen species ROS Sodium dodecyl sulfate SDS Tetramethylethylenediamine TEMED Trifluoroacetatic acid TFA
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