建構熱啟動 Taq 去氧核醣核酸聚合酵素及其突變蛋白之功能研究

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研究生：	楊家琪 Yang, Chia-Chi
論文名稱：	建構熱啟動 Taq 去氧核醣核酸聚合酵素及其突變蛋白之功能研究 Construction of a hot start Taq DNA polymerase and functional studies of its mutants
指導教授：	呂平江 Lyu, Ping-Chiang
口試委員:	藍忠昱 Lan, Chung-Yu 周裕珽 Chou, Yu-Ting
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2017
畢業學年度：	105
語文別：	英文
論文頁數：	76
中文關鍵詞：	熱啟動、去氧核醣核酸聚合酶、突變、建構蛋白
外文關鍵詞：	Hot start, Taq DNA polymerase, mutants, Construction
相關次數：	點閱：2 下載：0
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從嗜熱水生菌提取的去氧核醣核酸聚合酶 (Taq pol) 由於廣泛應用於 DNA 擴增、即時聚合酶鏈鎖反應 (real time PCR) 和 DNA 定序，目前已經成為分子生物學的常用試劑之一。傳統上大多使用 Pluthero 的方法萃取 Taq pol 但此純化方法需要耗費較長時間，因此已開發出利用大腸桿菌過表達重組的 Taq pol，並且在該系統中也研究了改善其功能的突變蛋白。因為 Taq pol 在室溫下具有顯著的活性，使聚合酶鏈鎖反應中形成非特異性產物影響後續實驗。一些研究顯示 Klentaq (N端缺失Taq pol) 在 37℃ 下可以顯著降低酵素活性，而在反應溫度下 (約72℃) 可維持正常活性。另外也有文獻顯示一些具有突變的 Klentaq 表現出更快或對抑制物有更高容忍度的性質。研究的目的是建構一個可以提供效率更佳的熱啟動Taq DNA聚合酶。在我們的研究中，首先構建了全長 Taq DNA聚合酶 (Taq pol) 和截短的 Taq DNA 聚合酶 (Klentaq，N端1-235個胺基酸缺失)以及數個文獻證實不同功能的單點或多點突變，以比較它們的活性。並利用即時螢光定量 PCR 檢測這些不同功能的突變蛋白，包含對溫度敏感性和提升擴增速率。結果顯示，結合熱啟動功能和增加延長能力點突變的 Taq pol 和 Klentaq 都具有熱穩定性。即時熒光定量PCR結果也顯示 Taq pol 和 Klentaq 上的 E742R/A743K 突變比野生型反應速度更快，並且同時在結合熱啟動和快速功能的多點突變體觀察到相同結果。而在溫度敏感性試驗中，含有 E626K 或 I707L 突變的 Taq pol 和 Klentaq 可在低溫下顯著降低活性並在 PCR 步驟中增加活性。這些結果表明了多個突變可以將不同的酵素性質組合在一起，也顯示我們可以藉由不同功能的酵素組合改進其功能並可在生活中更廣泛應用。

DNA polymerase from Thermus aquaticus (Taq pol) has become a common reagent in molecular biology because of its widely used in DNA amplification, quantitative real time polymerase chain reaction (real time PCR) and DNA sequencing. Most Taq pol was usually extracted with Pluthero's method, but the purification requires long time. Therefore, recombinant Taq pol from overexpression in Escherichia coli was developed, and in this system mutants improving its function were also studied. Taq pol has significant activity at room temperature, and then causes non-specific priming in PCR. Some reports showed that a truncated form of Taq pol, Klentaq (an N-terminal deletion), had apparently reduced activity at 37°C and retain normal activity at reaction temperature (about 72°C). The other reports said that Klentaq with some mutations revealed faster or inhibitor-resistant properties. The aim of the study is to create a hot start Taq DNA polymerase which can also provide better efficiency. In our study, several mutations on both full length Taq (Taq pol) and truncated Taq (Klentaq, deletion of N-terminal 1-235 amino acids) were constructed to compare their activities. Real time PCR was used to test the function of these mutants, and check points were temperature sensitivity and amplification. Not only that, we also focused on developing time saving method to produce Taq pol, Klentaq and their mutants. Results showed that both Taq pol and Klentaq which combined hot start function and increasing elongation ability mutation had heat stability. According to real time PCR results, mutant E742R/A743K on both Taq pol and Klentaq showed faster elongation rate than that of wild type. In temperature-sensitivity test, Taq pol and Klentaq which containing single mutation of E626K or I707L showed significantly decreasing activity at lower temperature and increasing activity at PCR step. Our results also revealed that multiple mutations could combine different properties. With the different enzyme function combination, multiple mutation on DNA polymerase allowed many extensive applications in our life.

中文摘要………………………………………………………………………………2
Abstract……………………………………………………………………………….3
謝誌……………………………………………………………………………………5
Contents……………………………………………………………………………….6
Contents of Tables and Figures………………………………………………………8
Chapter 1. Introduction…………………………………………………….……….10
1.1 Taq DNA polymerase………………………………………………….…….10
1.2 Hot start PCR……………………………………………………………..…10
1.3 Function analysis of truncation and different point mutations on
Taq DNA polymerase……….………………………………………….....…12
1.4 Motivation…………………………………………………………………..13
Chapter 2. Materials and Methods…………………………………………………15
2.1 Materials…………………………………………………………………….15
2.2 Protein construction, recombinant and mutagenesis of Taq pol……………...15
2.2.1 Construction of Klentaq polymerase……………………………………15
2.2.2 Construction of Taq pol and Klentaq mutants…………………………...16
2.3 Protein expression and purification………………………………………….16
2.3.1 Expression of Taq pol, Klentaq and their mutants………………………16
2.3.2 Purification of Taq pol, Klentaq and their mutants……………………...17
2.4 Identification of Taq pol, Klentaq and their mutants…………………………18
2.4.1 SDS-PAGE………………………………………………………...…...18
2.4.2 Western Blot…………………………………………………………….18
2.4.3 In-gel digestion and protein identification by MALDI-TOF/TOF MS….18
2.5 Quantification of protein concentration……………………………………..20
2.6 Polymerase Chain Reaction (PCR) assay……………………………………20
2.7 Functional Assays………………………………………………………...…21
2.7.1 Heat stability property…………………………………………………..21
2.7.2 Quantitative real time PCR assay……………………………………….21
2.7.3 Temperature sensitivity test……………………………………………..22
Chapter 3. Results and Discussions………………………………………………...23
3.1 Construction of Taq pol, Klentaq and their mutants………………………….23
3.2 Protein expression and purification………………………………………….24
3.3 Protein quantification and identification…………………………………….25
3.4 The enzyme activity of Taq pol, Klentaq and their mutants………………….26
3.5 Heat stability of Taq pol, Klentaq and their mutants…………………………27
3.6 Quantitative real time PCR assay……………………………………………28
3.7 Temperature sensitivity test of Taq pol, Klentaq and their mutants…………..30
Chapter 4. Conclusion………………………………………………………………33
Tables and Figures…………………………………………………………………..35
References…………………………………………………………………………...72
Appendix………………………………………………………………….…………76
 
Contents of Tables and Figures

Table 1. Mutagenized sites and their expected function………………………………35
Table 2. Oligonucleotides primers for constructing Klentaq and mutants…………….36
Table 3. Molecular weights and extinction coefficient of Taq pol, Klentaq
and their mutants…………………………………………………………….37
Table 4. DNA sequence and oligonucleotides primers for functional assay…………..38
Table 5. Mass list of Taq pol, Klentaq and their mutants………………………………39
Table 6. Summary of Taq pol, Klentaq and their mutants………...…………………...40
Figure 1. Construction of the recombinant plasmid…………………………………..41
Figure 2. PCR products of Klentaq insert and its mutants…………………………….42
Figure 3. The flow chart of protein expression and purification………………………44
Figure 4. Purification of Taq pol and its mutants……………………………………...45
Figure 5. Purification of Klentaq and its mutants……………………………………..47
Figure 6. Western blot of Taq pol and its mutants……………………………………..49
Figure 7. Western blot of Klentaq and its mutants…………………………………….50
Figure 8. The enzyme activity of Taq pol and its mutants……………………………..51
Figure 9. The enzyme activity of Klentaq and its mutants…………………………….52
Figure 10. Heat stability of Taq pol and its mutants…………………………………...53
Figure 11. Heat stability of Klentaq pol and its mutants………………………………55
Figure 12. TaqMan quantitative real time PCR assay of Taq pol and its mutants……...57
Figure 13. CT value of TaqMan quantitative real time PCR assay of Taq pol
and its mutants………………...………………………………………….58
Figure 14. SYBR Green quantitative real time PCR assay of Taq pol and its mutants...59

Figure 15. CT value of SYBR Green quantitative real time PCR assay of Taq pol
and its mutants…………………………………………………...……….60
Figure 16. SYBR Green quantitative real time PCR assay of Klentaq and its mutants..61
Figure 17. CT value of SYBR Green quantitative real time PCR assay of Klentaq
and its mutants…………………...……………………………………….63
Figure 18. Running method of temperature sensitivity test…………………………...64
Figure 19. Temperature sensitivity test of Taq pol and its mutants……………………65
Figure 20. CT value of temperature sensitivity test of Taq pol and its mutants………...66
Figure 21. Temperature sensitivity test of Klentaq and its mutants…………………...67
Figure 22. CT value of temperature sensitivity test of Klentaq and its mutants……...69
Figure 23. The mutational sites of Taq DNA polymerase……………………...……70

                                

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