藍藻蛋白 (Cyanophycin) 又稱為藻青素是一種天然來源的類蛋白質聚合物，其分子量可以從 25000 到 100000 道爾吞，在工業上可被用來製造聚天門冬胺酸 (polyaspartic acid)，而聚天門冬胺酸可被廣泛應用在許多工業、生物醫學與藥品製造方面。目前發現藍藻蛋白合成酶 (cyanophycin synthetase﹔EC 6.3.2.29) 是唯一可以催化合成藍藻蛋白的酵素，文獻指出藍藻蛋白合成酶的活性與 ATP、鉀離子、鎂離子或是硫醇化合物 (thiol reagent) 有關。
本實驗將源自藍細菌 Synechocystis sp. PCC6803 的藍藻蛋白合成酶，利用選殖的方法建構於 pRham N-His SUMO Kan Vector 上並形成重組載體 pRham-cphA，由於此載體本身在 N 端帶有六個組胺酸形成 His-tag，以利後續之純化，將此重組載體 pRam-cphA 轉型至 Escherichia coli NEB-turbo 中表現，在 0.1％ 的 rhamnose 下誘導 20 小時，有助於目標蛋白大量的表現，將目標蛋白質經過鎳親和性管柱純化可得到重組型藍藻蛋白合成酶，其分子量約為 98 kDa。
重組型藍藻蛋白合成酶其酵素特性如下，最適作用溫度為 55℃，最適作用 pH 值為 9.0 (使用 Tris-HCl 緩衝溶液)，於 20~50℃ 之間保溫 2 小時仍具有 80％ 以上的穩定性﹔於 25℃ 下在 pH 7.0〜9.5 (使用 Tris-HCl 緩衝溶液) 環境酵素仍可維持 70％ 以上的穩定性，此外，重組型藍藻蛋白合成酶需添加鎂離子才會具有活性，而以 L-精胺酸 (L-arginine) 和 L-天門冬胺酸 (L-aspartic acid) 共同作為基質時有最好的酵素活性。

Cyanophycin is a natural source protein-like polymer which has a molecular mass of 25 to 100 kDa，it can be used for the production of polyaspartic acid﹔Polyaspartic acid is used in a number of industrial﹐biomedical﹐and medical applications. Synthesis of cyanophycin is only catalyzed by cyanophycin synthetase﹐the activity of cyanophycin synthetase is generally depends on the presence of ATP﹐K+﹐Mg2+﹐and thiol reagent.
In this study﹐cyanophycin synthetase (cphA) was cloned into pRham N-His SUMO Kan Vector﹐and a recombinant plasmid pRham-cphA was be constructed. The plasmid with a N-terminal His-tag was transformed into Escherichia coli NEB turbo﹐then rhamnose (final concentration 0.1％) was added﹐and the cultivation was incubated for 24 hours. Cyanophycin synthetase was purified by nickel affinity chromatography﹐and SDS-PAGE analysis revealed that recombinant protein had approximately 98 kDa molecular mass.
The optimum activity of recombinant cyanophycin synthetase was obtained at 55 ℃ and pH 9.0 (Tris-HCl buffer). Recombinant cyanophycin synthetase remained more than 80％ residual activity after treatment with 20~50 ℃ for two hours and more than 70％ residual activity after incubated in pH 7.0~9.5 for 24 hours at 25 ℃. Recombinant cyanophycin synthetase had enzyme activity when Mg2+ were added. In substrate specificity test﹐recombinant cyanophycin synthetase had enzyme activity when using L-arginine and L-aspartic acid as substrate.

目錄

目錄 i
圖目錄 iv
表目錄 iv
附錄目錄 ivi
壹、前言 7
貳、文獻整理 7
1. 藍藻蛋白 (Cyanophycin) 簡介 7
1-1 藍藻蛋白的發現 7
1-2 藍藻蛋白的結構 8
1-3 藍藻蛋白的特性 8
1-4 藍藻蛋白的生產 8
1-5 藍藻蛋白的應用 9
2. 聚天門冬胺酸的生產方式與運用 10
3. 藍藻蛋白合成酶簡介 10
3-1 藍藻蛋白合成酶的發現 10
3-2 藍藻蛋白合成酶的序列分析 11
3-3 藍藻蛋白合成酶的特性 11
4. Synechocystis sp. PCC6803 菌株簡介 11
5. pET 表現系統簡介 12
6. Expresso® Rhamnose SUMO表現系統簡介 12
叁、實驗設計與流程 13
肆、實驗材料 14
1. 實驗菌株與載體 14
1-1 cphA基因來源 14
1-2 表現載體 14
1-3 實驗菌株 14
2. 實驗藥品 14
2-1 抗生素 14
2-2 蛋白質標準品 14
2-3 DNA 標準品 14
2-4 市售套組 14
2-5 酵素 14
2-6 化學藥品 14
3. 儀器設備 15
伍、實驗方法 17
1. 源自 Synechocystis sp. PCC6803 的 cphA 基因於 pET 系統之表現 17
1-1 製備小量質體 DNA 18
1-2 製備 DNA 洋菜糖膠體 17
1-3 DNA 濃度之定量 17
1-4 製備 E. coli DH5α 電穿孔勝任細胞 17
1-5 製備 E. coli DH5α 熱休克勝任細胞 18
1-6 電穿孔轉形作用 18
1-7 熱休克轉形作用 18
1-8 Colony PCR 篩選轉形株 18
1-9 洋菜膠電泳分析 19
1-10 序列分析 19
1-11 以 Dpn1 酵素剪切具甲基化的模板雙股 DNA 19
1-12 菌種保存 19
2. 源自 Synechocystis sp. PCC6803 的 cphA 基因於 pRham 系統之表現 19
2-1 製備小量質體 DNA 18
2-2 DNA 濃度之定量 19
2-3 Synechocystis sp. PCC6803-cphA 基因之擴增 19
2-4 洋菜膠電泳分析 20
2-5 熱休克轉形作用 20
2-6 Colony PCR 篩選轉形株 20
2-7 洋菜膠電泳分析 20
2-8 序列分析 21
2-9 菌種保存 21
3.重組型 Synechocystis sp. PCC6803 藍藻蛋白合成酶之蛋白質表現與純化 21
3-1 測試不同濃度 IPTG 誘導蛋白質表現 21
3-2 測試不同濃度 rhamnose 誘導蛋白質表現 21
3-3 重組型 Synechocystis sp. PCC6803 藍藻蛋白合成酶於大腸桿菌中之表 現 21
3-4 重組型 Synechocystis sp. PCC6803 藍藻蛋白合成酶之純化 21
3-5 蛋白質定量 22
3-6 蛋白質電泳分析 22
3-6-1 SDS-PAGE蛋白質電泳藥品 22
3-6-2 SDS-PAGE蛋白質電泳膠體的製備 23
3-6-3 SDS-PAGE蛋白質電泳之操作方法 24
3-6-4 膠片染色與脫色 24
4. 重組型 Synechocystis sp. PCC6803 藍藻蛋白合成酶的特性探討 24
4-1 活性探討 24
4-1-1 Malachite Green 溶液配置 24
4-2 最適作用溫度探討 24
4-3 最適作用pH 值探討 25
4-4 酵素熱穩定性探討 25
4-5 酵素 pH 值穩定性探討 25
4-6 金屬離子與化學藥劑之影響 25
4-7 基質特異性 25
陸、結果與討論 26
1. 源自 Synechocystis sp. PCC6803 的 cphA 基因於 pET 系統之表現 26
2. 源自 Synechocystis sp. PCC6803 的 cphA 基因於 pRham 系統之表現 26
3. 重組型Synechocystis sp. PCC6803-cphA 之蛋白質表現及純化 26
4. 重組型Synechocystis sp. PCC6803-cphA 之特性分析 28
柒、結論 31
捌、參考文獻 32

圖目錄
圖一、以 SDS-PAGE 分析含有 pET21b(+)-cphA plasmid 之 E. coli BL21(DE3)-CodonPlus-RIL 以不同 IPTG 濃度誘導之總蛋白分布................................................................................................................

44
圖二、以洋菜糖膠體電泳分析 Synechocystis sp. PCC6803 –cphA 基因 PCR 擴增之產物......................................................................................
45
圖三、以洋菜糖膠體電泳分析 colony PCR 擴增之產物.................................. 46
圖五、以 SDS-PAGE 分析含有 pRham-cphA 之 E. coli NEB turbo 以不同 rhamnose 濃度誘導之總蛋白分布..........................................................
47
圖六、藍藻蛋白合成酶純化過程蛋白質分布情形之 SDS-PAGE 膠體電泳圖............................................................................................
48
圖七、重組型 Synechocystis sp. PCC6803 藍藻蛋白合成酶最適作用溫度之探討..........................................................................................................
49
圖八、重組型 Synechocystis sp. PCC6803 藍藻蛋白合成酶最適作用 pH 之探討............................................................................................................
50
圖九、重組型 Synechocystis sp. PCC6803 藍藻蛋白合成酶之熱穩定性................................................................................................................
51
圖十、重組型 Synechocystis sp. PCC6803 藍藻蛋白合成酶之 pH 穩定性................................................................................................................
52












表目錄
表一、 Synechocystis sp. PCC6803-cphA 基因轉殖及 colony PCR 使用之引子序列分析...............................................................................................
53
表二、 重組型 Synechocystis sp. PCC6803 藍藻蛋白合成酶之純化表........ 54
表三、 金屬離子與化學物質對重組型 Synechocystis sp. PCC6803 藍藻蛋白合成酶活性之影響.............................................................................
55
表四、 重組型 Synechocystis sp. PCC6803 藍藻蛋白合成酶之基質特異性.. 56

 

附錄目錄
附錄一、 藍藻蛋白的構造與化學式................................................................... 37
附錄二、 電子顯微鏡下的細胞包涵體................................................................ 38
附錄三、 聚天門冬胺酸的工業合成法................................................................ 39
附錄四、 藍藻蛋白的生物降解過程................................................................... 40
附錄五、 pET 蛋白質表現系統........................................................................... 41
附錄六、 Expresso® Rhamnose SUMO system 流程示意圖..............................


附錄七、 pRham N-His SUMO Kan 質體示意圖……………........................... 42


43

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