1.蛋白質的取得：
首先透過 PCR 與 DNA 定序的方式獲得保存菌種的 DNA 序列，再利用 NCBI 網站做DNA 序列的 Blast，確認前人保存的菌種是表現 MTGFP 重組蛋白質的菌株。
再透過E.coil 表現 MTGFP 蛋白質，並用 Western 做抗體專一性的確認。隨後再利用 E.coil 大量培養，並利用破菌及清洗蛋白質的方式，獲得大量 MTGFP 蛋白質的內涵 體，雖然此時MTGFP 純度高但不具有生物活性，所以 MTGFP 蛋白質利用階段性熱 平衡透析法，將摺疊法蛋白質回復到自然態，進行後續的各項實驗。
2.MTGFP 的蛋白質摺疊：
觀察本實驗所使用的蛋白質摺疊法為階段性熱平衡透析法，也就是利用凖靜態 的過程來進行蛋白質的透析與摺疊，除了可以避免蛋白質因外界環境劇烈變化而沈 澱，也可以避免蛋白質彼此交互作用而聚集，使蛋白質分子能很有效率進行摺疊達 到自然態。而自然態的蛋白質其定義不僅只有結構回到自然態，並且也要具有生物 活性才算成功的蛋白質復性。所以本實驗收集每個溶液在不同階段下的蛋白質樣品 及置換過後的緩衝溶液，測量這些摺疊中間體，摺疊過程中其結構及功能的變化。
在結構分析的方面，1.利用圓二色光譜儀 (CD) 測量蛋白質的二級結構的比例，得 知GFP 在一開始時其結構並沒有完全解開，但與隨著摺疊的過程，使 MT 蛋白質得 結構逐漸形成；2.利用動態光散射儀(DLS)測量蛋白質在摺疊過程中，蛋白質分子逐 漸摺疊接近自然態，其蛋白質粒徑也逐漸縮小；3.利用螢光儀分析蛋白質內的芳香族 胺基酸的螢光變化，由於蛋白質中的苯環胺基酸會因為吸收波長280 nm 的紫外光，
而造成苯環的電子共振產生螢光。然而利用波長280 nm 的激發光偵測蛋白質的摺疊 狀態時，發現蛋白質釋放出的螢光強度隨著摺疊越接近自然態而越增加，且其螢光 波峰也逐漸往短波長移動，就是俗稱的藍位移，也就表示蛋白質因為摺疊步驟接近 自然態時，其蛋白質的摺疊結構比較緊密，此時疏水性胺基酸通常已經被包埋在蛋 白質內部形成疏水性的核心形成穩定結構，所以能量不易被釋放，因此可使得螢光 強度較強也較高。反之，蛋白質在摺疊初期疏水性胺基酸暴露在親水性的環境中，
所以蛋白質結構很不穩定，會和環境周圍的粒子作用產生能階不穩定，使得能量以 熱能的形式流失，因此螢光的能量也較低，朝向長波長移動就是俗稱的紅位移。綜 合以上三種方式可以確認MTGFP 蛋白質在結構上已經摺疊回自然態。
在功能方面，因為 MTGFP 是個融合蛋白，所以仍然保留原本蛋白質分子的特
性，利用ICP-AES 偵測 MTGFP 蛋白質上有七個金屬螯合位置對金屬的螯合能力，
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然而測量後發現幾乎每個 MTGFP 分子，可接 6~7 個金屬；再利用螢光蛋白激發 p–hydroxybenzyliden-imidazolidinone 的結構後，可產生能量共振釋出螢光。
在本論文中發現兩個特別的現象，第一：MTGFP 在蛋白質摺疊過程中，利用 395 nm 觀察 MTGFP 分子內的 GFP 時，其 GFP 螢光的強度隨著蛋白質摺疊過程而逐 漸降低(圖 31)，可能是在蛋白質摺疊過程中 GFP 會把能量轉給 MT，產生 Quenching 效應；第二：用3 D 螢光光譜分析純 MT 及純 GFP，發現這兩個蛋白質其螢光基團 彼此很相近(圖 34、圖 35)，所以在本實驗室自行合成的 MTGFP 蛋白質分子內，MT 除了有可能是Quencher 抵制 MTGFP 中的 GFP 螢光外，也有可能 MT 可以產生 FRET 的現象將能量轉給GFP。
為了要證明MTGFP 中的 MT 有螢光轉移的效應的產生，將能量轉給 GFP，於 是利用激發光350 nm 觀察 MTGFP 中的 MT 蛋白質，在扣除掉 GFP 影響因素下，其 螢光強度低於相同濃度的 MT 蛋白質(圖 38)。再者利用酵素(Enterokinase)切割此分 子內的MT 及 GFP，進行跑 Native page 確認螢光蛋白的分佈，並利用波長 300 nm 的紫外光照膠臺去偵測蛋白質螢光，發現 MTGFP 蛋白質的螢光隨著酵素增加，使 得 MT 及 GFP 的距離增加，然而蛋白質螢光隨之上升，所以推測 MT 蛋白質的結 構會吸收GFP 的螢光(圖 39、圖 40)。然而 MTGFP 蛋白質在摺疊過程中其 GFP 的能 量有逐漸降低，但根據能量不滅原理，這些喪失的能量應該是以熱能的形式釋出。
總結:我們發現 MT 蛋白質本身具有金屬環，可以產生 FRET 效應，將螢光能量 轉給 GFP，同時也是可以將 GFP 的螢光能量消耗的殺手，因為隨著蛋白質摺疊結構 越來越接近 native state 時，MT 蛋白質會變成 quencher 走static quenching 路徑 quench GFP 蛋白質螢光的表現。另外對於未來發展，MT 本身是 random coil 的形式，
沒有特定結構，所以未來可以利用 MTGFP 蛋白質，做為 MT 蛋白質的摺疊即時的 偵測器。
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