臺灣博碩士論文加值系統

大腸桿菌表現系統是目前生產異源蛋白的最佳途徑，但瓶頸是有時會在胞內形成了包涵體。大量的重組蛋白中因而出現不可溶的部分，此問題可利用8 M尿素或6 M鹽酸胍之變性劑溶液，將包涵體溶解之，使變性的蛋白質重新摺疊，恢復原來蛋白質的結構與活性。
重組型幾丁質酶AsChi61產物中以N-乙醯幾丁六醣活性最高且具有SDS耐受性，其結構較為堅固，在工業應用上不易被環境因子影響。在過去研究中可知N-乙醯幾丁六醣對於免疫反應與抗腫瘤是有相關的。重組型幾丁質酶AsChi61可符合所考慮之因素包含了高產量、生產時間短及製程簡易，可使其適用於工業應用並量產之。
本研究利用pET15b表現系統來生產出重組型幾丁質酶AsChi61，經SDS-PAGE電泳分析，篩選出誘導表現良好的菌株，再進行大量表現，但過程中有包涵體形成的情形，利用不同濃度之尿素，使胞內中的包涵體變性後再重新摺疊，使重組型幾丁質酶的表現活性能夠提升，並以鈷樹脂將其純化之。

The main host for over-expression of recombinant proteins in prokaryotic system is Escherichia coli. However, one of the major obstacles in E. coli is the recombinant in inclusion body formation. Then the target is an insoluble form. The strong protein denaturant such as 8 M urea or 6 M guanidinium hydrochloride is applied to denature the structure of protein for solving in an aqueous solution. Finally, urea or guanidinium hydrochloride is removed gradually by dialysis to allow refolding of the denatured proteins.
The chitinase, AsChi61, possess high chitolytic activity and SDS resistance. This indicates its structure is too rigid to tolerate the environmental factors when applied in industry. In addition, the major products obtained from chitin digested by AsChi61 are N-acetamidine hexahexose. Hexameric oligochitin was known to be immune enhancer with anti-tumor activity. The reAsChi61 with those advantages are expressed in inclusion body in Escherichia coli system.
In this study, reAsChi61 was expressed in the pET15b system, and then analyzed on SDS-PAGE electrophoresis. In the purification, urea was applied to solve the protein. With different concentration of urea, some inclusion bodies were denatured. However, the His-tag of reAsChi61 was still bound to the resin. After elution, the expression activity and amount of the recombinant chitinase can be improved.

明志科技大學碩士學位論文指導教授推薦書 i
明志科技大學碩士學位論文口試委員審定書 ii
誌謝 iii
中文摘要 iv
Abstract v
目錄 vi
圖目錄 xi
表目錄 xii
縮寫對照表 xiii
第一章 緒論 1
第二章 文獻回顧 3
2.1 幾丁質及其衍生物 3
2.2.1 幾丁質(chitin) 3
2.1.2 幾丁聚醣(chitosan) 3
2.2 幾丁質結構 4
2.2.1 化學結構 4
2.2.2 晶體結構 4
2.3 幾丁質在自然界的分佈 5
2.3.1 節肢動物 5
2.3.2 軟體動物 5
2.3.3 腔腸動物 5
2.3.4 海藻類 6
2.3.5 真菌類 6
2.4 幾丁質及其衍生物的製備 6
2.4.1 化學修飾法 7
2.4.2 超音波降解法 7
2.4.3 酸水解法 7
2.5 幾丁質酵素 7
2.5.1 幾丁質酵素 7
2.5.2 動物幾丁質酵素 8
2.5.3 植物幾丁質酵素 8
2.5.4 微生物幾丁質酵素 8
2.6 幾丁質酶之命名 9
2.6.1 Family 18 9
2.6.2 Family 19 9
2.6.3 Family 20 10
2.7 幾丁質酶水解模式分類 10
2.7.1 內切型幾丁質酶(endo-chitinase) 10
2.7.2 外切型幾丁質酶(exo-chitinase) 10
2.7.3 任意型幾丁質酶(random chitinase) 11
2.7.4 轉醣型幾丁質酶(transglycosylation-catalyzingchitinase) 11
2.8 幾丁質酶之純化 11
2.8.1 野生型幾丁質酶純化 11
2.8.2 重組型幾丁質酶純化 11
2.8.2.1 His-tag重組蛋白質純化 12
2.8.2.2 GST融合重組蛋白質純化 12
2.9 幾丁質酵素活性分析方法 12
2.9.1 還原醣測定法 12
2.9.2 DNS測定法 13
2.9.3 幾丁質藍測定法(chitin azure) 13
2.9.4 膠態幾丁質藍測量法(colloidal chitin azure) 13
2.9.5 CM-chtitn-RBV測量法 13
2.9.6 N-乙醯葡萄糖胺生成量測定法 14
2.9.7 濁度計測定法 14
2.9.8 黏度計測定法 14
2.9.9 螢光光度測定法 14
2.9.10 電子掃描測定法 14
2.10 以大腸桿菌表現重組蛋白質的系統 15
2.10.1 pET表現系統 15
2.10.2 包涵體(inclusion bodies) 15
2.11 蛋白質的鑑定 15
2.11.1 質譜法 15
2.11.2 蛋白質之N端胺基酸定序 16
2.12 幾丁質酵素AsChi61 16
2.12.1 於pET表現系統表現 17
第三章 實驗規劃 19
3.1 實驗目的及方法 19
3.2 實驗器材 20
3.2.1 儀器 20
3.2.2 藥品 21
3.2.3 材料 22
3.3 實驗方法 23
3.3.1 菌種來源 23
3.3.2 dTB粉末 23
3.3.3 dTB(amp)培養液配製 23
3.3.4 dTB(amp)培養基配製 23
3.3.5 LB(amp)培養液配製 23
3.3.6 轉型(transformation) 24
3.3.7 液態培養 24
3.3.8 IPTG誘導重組蛋白生產 24
3.3.9 小量表現(small-scale expression) 24
3.3.9.1 樣品取樣 24
3.3.10 SDS-PAGE 24
3.3.10.1 鑄膠玻璃板之裝備 25
3.3.10.2 濃度10%分離性膠體之配製 25
3.3.10.3 堆積性膠體之配製 25
3.3.10.4 蛋白質電泳操作 26
3.3.10.5 蛋白質電泳膠體CBB染劑 26
3.3.10.6 蛋白質電泳膠體CBB染色 26
3.3.10.7 蛋白質電泳膠體CBB褪染 26
3.3.11 蛋白質大量表現(large scale protein expression) 26
3.3.11.1 試劑製備 26
3.3.11.1.1 濃度10 mg/ml溶菌酶之配製 26
3.3.11.1.2 濃度10 mg/ml DNase I之配製 26
3.3.11.1.3 濃度10 mg/ml RNase A之配製 27
3.3.11.1.4 磷酸緩衝液(PB)之配製 27
3.3.11.1.5 磷酸緩衝生理食鹽水溶液(PBS)之配製 27
3.3.11.1.6 咪唑(imidazole)沖提液之配製 27
3.3.11.2 pET系統大量表現 28
3.3.11.2.1 菌體發酵 28
3.3.11.2.2 樣品製備 28
3.3.11.2.3 樣品純化 28
3.3.12 以凝血酶水解His-tag 28
3.3.13 蛋白質轉漬法 29
3.3.13.1 試劑製備 29
3.3.13.1.1 CAPS轉漬緩衝液 29
3.3.13.1.2 轉漬染色液 29
3.3.13.1.3 轉漬褪色液 29
3.3.13.2 轉漬操作程序 29
3.3.14 蛋白質定量 29
第四章 實驗結果 31
4.1 菌種培養 31
4.2 於pET系統重組蛋白AsChi61 31
4.2.1 小量表現 31
4.2.2 大量表現 31
4.2.3 以尿素純化包涵體之重組蛋白質 31
4.2.4 重組蛋白質AsChi61純化後以凝血酶水解 32
4.2.5 重組蛋白質之濃度 32
第五章 討論 33
第六章 未來展望 34
第七章 參考文獻 56

圖目錄
圖一、纖維素、幾丁質、幾丁聚醣之化學結構 35
圖二、自然界中常見幾丁質之晶體型態結構 36
圖三、α-chitin與β-chitin的分子鏈排列情形 37
圖四、幾丁質酶之四種水解模式 38
圖五、蛋白質正確摺疊與錯誤摺疊和聚合的簡化模型 39
圖六、pET15b質體 40
圖七、表現載體pET15b轉型至大腸桿菌(BL21)之三區劃線 41
圖八、表現載體pET15b-AsChi61轉型至大腸桿菌(BL21)之三區劃線 42
圖九、以IPTG誘導大腸桿菌BL21(△DE3)在胞內產出重組蛋白質AsChi61 43
圖十、純化大量表現重組蛋白質pET15b-AsChi61凝膠電泳 44
圖十一、以尿素純化pET15b-AsChi61中包涵體之凝膠電泳 45
圖十二、以凝血酶水解純化後pET15b-AsChi61之凝膠電泳 46
圖十三、BSA標準曲線製備 47

表目錄
表一、各濃度的SDS-PAGE製作劑量表 48
表二、SDS-PAGE電泳試劑配置表 49
表三、dTB固態培養基配置表 50
表四、dTB培養液配置表 51
表五、LB培養液配置表 52
表六、CBB染色液及褪染液配置表 53
表七、蛋白質定量 54
表八、AsChi61之胺基酸序列 55

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