臺灣博碩士論文加值系統

直到現在，T7 基因表現系統仍是最常用來以大腸桿菌生產重組蛋白質的一個方法，不過由於這個系統需仰賴使用化學藥物IPTG來誘導，而此化學藥物價格偏高且有毒性，因此限制了這個系統在工業界上的應用性。在本研究中，我們將這個系統修正為可以用溫度或是使用阿拉伯糖來誘導。系統的建構則首先將受lPRPL 或 araBAD啟動子分別調控T7 RNA聚合酉每基因表達的DNA片段箝入大腸桿菌染色體的lacZ基因位置中，接著為了降低目標基因在質體上未誘導的表達量，我們在質體上建構了兩種溫度敏感性的lacI基因，包括在lacI基因中的187胺基酸由glycine改為serine（Gly187→Ser187）和265胺基酸由glycine改為aspartate（Gly265→Asp265），最後為了避免野生型和圖變型lacI基因互相干擾，菌種染色體上的lacI基因也予以剔除。
為了進一步檢測建構系統的特性，我們選擇LacZ蛋白質作為生產模型。經過實驗測試，建構有溫度調控T7 基因表現系統和含有溫度敏感性lacI基因（Gly265→Asp265）重組質體的菌種，當培養溫度由30℃升高到39℃時，可以生產58,000 Miller units的LacZ蛋白質；而當培養溫度提升到40℃時，建構有溫度調控T7 基因表現系統和含有溫度敏感性lacI基因（Gly187→Ser187）重組質體的菌種則能生產46,000 Miller units的LacZ蛋白質。相較於未受溫度誘導菌種的LacZ蛋白質產量，這些蛋白質產量估計增加了約200倍。至於使用建構有阿拉伯糖調控T7 基因表現系統的菌種，LacZ蛋白質生產的狀況類似於建構有溫度調控T7 基因表現系統的菌種。
為了瞭解T7 RNA聚合酉每基因箝入在大腸桿菌染色體中的位置對於T7 基因表現系統之影響，我們將受lPRPL啟動子分別調控T7 RNA聚合酉每基因表達的DNA片段箝入大腸桿菌染色體的rha基因位置中。隨後以LacZ蛋白質作為生產模型，結果顯示這樣建構的菌種生產LacZ蛋白質的趨勢大致與上述的菌種相似，但是蛋白質生產量均降低，這也意謂著T7 基因表現系統的效能確會受到T7 RNA聚合酉每基因箝入在大腸桿菌染色體中的位置之影響。
實際上，運用受溫度調節的T7 基因表現系統來生產重組蛋白質特別吸引人，因為其誘導的方式兼具有簡單操作、經濟性和有效性等特質。因此我們選擇了aspartase作為生產的酵素，來檢測這套系統對於生產工業用酵素的可行性。經過測試得知，當培養溫度由30℃升高到37℃時，建構有溫度調控T7 基因表現系統和含有溫度敏感性lacI基因（Gly265→Asp265）重組質體的菌種可以生產高達58 U/mg單位活性的aspartase，而這個蛋白質產量估計佔了所有細胞蛋白質含量的20%。有趣的是，我們發現生產aspartase的菌種比未生產的菌種生長狀況佳，在本研究中，針對這個現象我們也提出了實驗證據並據以多方面討論。根據以上諸多實驗結果可以獲得一個結論，就是這個受溫度調節的T7 基因表現系統極具有工業生產重組蛋白質的實用價值。

The T7 expression system is by far the most commonly used system or producing recombinant proteins in Escherichia coli. However, the use of IPTG to activate this system has limited its practical applications in industry simply because of the high cost and inherent toxicity of this chemical. In this study, we have modified the T7 expression system become controllable by heat or L-arabinose. The gene encoding T7 RNA polymerase fused with either lPRPL or araBAD promoter was inserted into E. coli chromosome at the lacZ locus. To lower the basal transcript of target genes on the plasmid, the heat-labile lacI gene (lacIts) including Gly187→Ser187 and Gly265→Asp265 mutations was introduced into the plasmid construct with the transcriptional orientation divergent from the T7 promoter. Moreover, the lacI gene in E. coli chromosome was also removed to ensure no interference of wild-type lacI with the mutant type.
To examine the production characteristics of the constructed systems, the lacZ gene was chosen as a production model. With the use of the heat-regulated T7 system, the cell harboring the plasmid with inclusion of lacIts (Gly265→Asp265) enabled to produce LacZ of 58,000 Miller units pon receiving a temperature shift from 30 to 39oC. As the temperature was raised to 40 oC, the LacZ production accounting for 46,000 Miller units was obtained for the cell carrying the plasmid with the lacIts (Gly265→Asp265). These production yields were estimated to gain an around 200-fold increase in comparison with the uniduced state. Similar results were obtained for the cells equipped with L-arabinose-regulated T7 system.
To see how the insertion site may affect the efficiency of the system, the gene encoding T7 RNA polymerase fused with the lPRPL promoter was inserted into E. coli chromosome at the rha locus. Using the LacZ production model, similar but lower production profiles were obtained as compared to the cell with the insertion of the T7 RNA polymerase gene into lacZ. It indicates that the insertion site on the chromosome could have an impact on the performance of the T7 expression system.
The heat-controllable T7 expression system is particularly attractive to recombinant protein production because the induction method is simple, economic, and efficient. To demonstrate the feasibility of this system for high production of industrial enzymes, the aspA gene encoding aspartase was used. Upon shifting the temperature to 37 oC, the cell carrying the recombinant plasmid with lacIts (Gly265→Asp265) produced aspartase with 58 U/mg, and the protein production accounted for over 20% total cell protein content. Interestingly, the growth of aspartase-producing cells was not inhibited but improved instead as compared to the cell without aspartase production. The underlying reasons were also discussed. In conclusion, the T7 expression systems reconstructed to become thermo controllable show a grate promise for its applicability in industry.

第一章 緒論
1.1前言…………………………………………………………… 1.
1.2文獻回顧..…………………………………………………….. 2.
1.3研究動機…………………………………………………….... 8.
第二章 實驗方法
2.1 菌種之儲存與馴養…………………………………………… 12.
2.1.1 菌種儲存…………………………………………………... 12.
2.1.2 菌種馴養…………………………………………………... 12.
2.2 .DNA 純化方法……………………………………………….. 13.
2.2.1 質體純化………………………………………...………… 13.
2.2.2 瓊脂凝膠萃取DNA片段…….………………………….... 15.
2.2.3 .PCR DNA 純化…………………………………………… 15.
2.3 染色體純化方法……………………………………………… 16.
2.4 剪切反應、連接反應、凝膠電泳法………………………… 17.
2.4.1 剪切反應…………………………………………………... 17.
2.4.2 凝膠電泳法………………………………………………... 18.
2.4.3 連接反應…………………………………………………... 19.
2.5 酒精沈澱……………………………………………………… 19.
2.6. 勝任細胞的準備………………………………………………. 20.
2.6.1 化學法………………………………………………………. 20.
2.6.2 . 電擊穿透法………………………………………………... 21.
2.7轉殖作用(Transformation)……………………………………… 22.
2.7.1以化學法製備勝任細胞的轉殖…………………………….. 22.
2.7.2以電擊穿透法製備勝任細胞的轉殖……………………….. 22.
2.8. 轉導作用(Transduction)……………………………………….. 23.
2.8.1 P1vir 溶胞產物製備(Preparation of P1vir lysate)…….……. 23.
2.8.2以P1vir溶胞產物進行轉導作用…………………………... 24.
2.9 重組菌重製備………………………………………………….. 25.
2.10 聚合酵素連鎖反應(Polymerase Chain Reaction)……………. 25.
2.11重組菌種建構…………………………………………………. 27.
2.11.1剔除染色體lacI基因……………………………………… 27.
2.11.2插入式質體(integration vector)之建構……………………. 30.
2.11.3建構菌種 RY2405(△lacI)、RY2405(lacI42::Tn10)……... 34.
2.11.4建構菌種 RY2405(△lacI lacZ::lG2)、RY2405(lacI42::Tn10 lacZ::BAD)、RY2405(rha::lG2)…………………………. 35.
2.11.5建構VJS632(△lacI lacZ::lG2)、VJS632(lacI42::Tn10 lacZ::BAD)、VJS676(rha::lG2)…………………………. 40.
2.11.6建構質體pET-20bI、pET-187、pET-265
2.11.7建構質體 pKF3-Z[N]……………………………………... 42.
2.11.8建構質體 pET20b(+)-Z[N]、pET-20bI-Z[N]、 pET-187-Z[N]、pET-265-Z[N]…………………………… 43.
2.12系統檢測………………………………………………………. 45.
2.12.1蛋白質定量……………………………………………….. 45.
2.12.2 AspA酵素活性分析……………………………………… 45.

2.12.3蛋白質電泳(SDS-PAGE)…………………………………. 47.
2.12.4 .b-galactosidase 酵素活性分析………………………….. 49.
第三章 結果………………………………………………………... 51.
3.1系統簡述………………………………………………………. 51.

3.1.1簡述重組菌種特性………………………………………… 51.
3.1.2簡述所建構載體特性……………………………………… 54.
3.2 利用溫度來調控T7基因表達系統………………………….. 56.
3.2.1檢視菌種VJS632(△lacI lacZ::lG2)生產AspA酵素的特性…………………………………………………………... 56.

3.2.2初測目標蛋白質(AspA protein)之活性…………………… 62.
3.3 利用L-arabinose和溫度調控系統表達……………………... 64.
3.4利用溫度調控菌種VJS632(△lacI lacZ::λG2)來生產LacZ 81.
3.5檢測菌種VJS676(rha::lG2)生產LacZ之特性…..………….. 96.
3.6初步推測AspA對細胞生長的關係..……….………………… .110.
第四章 結論與未來展望.……………………………….…….…. 125.

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41. 沈仲澤 2000 發展以溫度方式來調控製造重組蛋白質 私立逢甲大學 化學工程研究所論文