Cloning, expression and characterization of a family B DNA polymerase from the hyperthermophilic archaeon thermococcus celericrescens and its application to PCR

국문 초록 (Abstract)

Thermococcus celericrescens 유래의 DNA polymerase (Tcel DNA polymerase) 유전자를 클로닝하고 염기 서열을 밝혔다. Tcel DNA polymerase 유전자는 775 개의 아미노산 잔기를 암호화하는 2,328 bp의 염기서열로 구성되어 있다. 다른 고세균 유래의 family B DNA polymerase의 서열과의 비교하였을 때, Pyrococcus kodakaraensis KOD1는 92%, Thermococcus celer는 86%, Pyrococcus furiosus는 79%의 상동성을 보였다. 단백질의 분자량은 90,134 Da이었다. Tcel DNA polymerase는 pET-20b(+) 와 Escherichia coli Rosetta (DE3) pLysS에서 발현되었으며, 열처리와 HisTrapTM HP 컬럼 크로마토그래피, HiTrapTM SP HP 컬럼 크로마토그래피를 거쳐 정제하였다. 특성 확인 결과, pH 8.0 와 70 °C에서 각각 최고의 활성을 보였다. Tcel DNA polymerase를 이용한 최적의 PCR buffer는 30 mM Tris-HCl (pH 8.4), 1.5 mM MgCl2, 60 mM KCl, and 0.0025% BSA였다. Tcel DNA polymerase의 error-rate와 extension efficiency들은 Taq과 Pfu DNA polymerase와 비교하여 측정하였다. Taq 과 Tcel DNA polymerase를 10:1 비율로 섞은 Tcel plus DNA polymerase를 사용하였을 때 가장 효율적으로 30 kb 까지 long range PCR이 가능하였다. 또한 두 개의 아미노산 잔기를 치환하여 야생형의 DNA polymerase보다 thermostability와 PCR effeciency가 향상된 돌연변이 Tcel DNA polymerase를 발굴하고자 하였다. 752번과 213번 잔기를 타켓으로 하여 점 돌연변이를 일으킨 결과, 세 종류의 돌연변이 Tcel DNA polymease에서 모두 thermostability와 PCR efficiency가 향상된 것을 확인하였다. 세 종류의 돌연변이 중에서 가장 PCR efficiency가 향상된 이중 돌연변이 TcelA752K/N213D DNA polymerase는 3' → 5' 엑소뉴클아제 활성에 영향을 미치는 ExoII 모티브에 돌연변이화 한 것임에도 불구하고 야생형의 Tcel DNA polymerase와 비교하여 3' → 5' 엑소뉴클아제 활성에 변화가 거의 없음을 확인 하였다. 이중 돌연변이 TcelA752K/N213D DNA polymerase는 Taq DNA polymerase보다 2.6배 향상된 충실도를 나타내었고, 더욱이 PCR 증폭 효율에 있어서는 2분의 증폭시간에도 10 kb의 긴 표적산물을 증하였다. 이러한 결과는 이중 돌연변이 TcelA752K/N213D DNA polymerase가 LA (long and accurate) PCR에 적용가능함을 보여주었다.

다국어 초록 (Multilingual Abstract)

The family B DNA polymerase gene from the euryarchaeon Thermococcus celericrescens (Tcel) contains a open reading frame of 2,328 bp that encodes 775 amino acid residues. In this study, the Tcel DNA polymerase gene was expressed in Escherichia coli strain Rosetta(DE3)pLysS. The expressed enzyme was purified by heat treatment, HisTrapTM HP column chromatography and HiTrapTM SP HP column chromatography and then characterized. The molecular mass of the purified Tcel DNA polymerases are about 90 kDa, as estimated by SDS-PAGE. The optimal pH and temperature of the purified enzyme were determined to be 8.0 and 70℃. The enzyme was activated by magnesium ion. The optimal buffer for PCR with Tcel DNA polymerase consisted of 30 mM Tris-HCl (pH 8.4), 1.5 mM MgCl2, 60 mM KCl, 0.0025% BSA. The error-rate and extension efficiency of Tcel DNA polymerase were tested and compared with Taq and Pfu DNA polymerases. Use of different ratios of Taq and Tcel DNA polymerases determined that a ratio of 10:1 efficiently facilitated long PCR (approximately 30kb). Also mutant DNA polymerases were created by site-directed mutagenesis targeting two residues to enhance thermostability and PCR efficiency than wild-type Tcel DNA polymerase. As the result of the point mutation by targeting residues of A752 and N213, it was confirmed that the PCR efficiency and thermostability were all enhanced with three of mutant Tcel DNA polymerases. Among these type of mutant, The double mutant TcelA752K/N213D DNA polymerase, enhanced the most PCR efficiency, was confirmed there was little change in 3' → 5' exonuclease activity comparing with wild-type Tcel DNA polymerase even though mutation in ExoII motif. The double mutant TcelA752K/N213D DNA polymerase had a 2.6-fold lower error rate than Taq DNA polymerase. Furthermore it could amplify the long PCR product of 10 kb even in extension time of 2 minute at PCR efficiency. It seems that the double mutant TcelA752K/N213D DNA polymerase can be apply to LA(long and accurate) PCR.

목차 (Table of Contents)

• Ⅰ. Introduction 1
• Ⅱ. Materials and Methods 3
• 1. Strains and culture conditions 3
• 2. Genomic DNA isolation 4
• 3. Sequence analysis 4
• 3.1 Consensus PCR 4
• 3.2 Primer walking PCR 5
• 3.3 DNA sequencing and sequence analysis 6
• 4. Construction of plasmid and expression of the Tcel DNA polymerase gene 7
• 5. Purification of Tcel DNA polymerase 8
• 6. Activity assay of Tcel DNA polymerase 9
• 6.1 Determination of optimum pH 10
• 6.2 Determination of optimum temperature 10
• 6.3 Determination of optimum concentration of MgCl2 and KCl 10
• 6.4 Thermostability 10
• 7. Exonuclease activity 11
• 8. Application to PCR amplification 11
• 8.1 Optimal condition of PCR using Tcel DNA polymerase 11
• 8.2 Long-range PCR of Tcel and Tcel plus DNA polymerases 13
• 8.3 Application of Tcel and Tcel plus DNA polymerases in time-saving PCR 13
• 9. Site-directed mutagenesis and plasmid construction 14
• 10. Expression and purification of mutant DNA polymerases 15
• 11. Assay for thermostability 16
• 12. Assay for PCR efficiency 16
• 12.1 Optimization of PCR amplification using mutant DNA polymerases 16
• 12.2 Time-saving PCR 16
• 12.3 Long-range PCR 17
• 13. Assay for fidelity 17
• Ⅲ. Results and Discussion 19
• 1. Sequence analysis 19
• 1.1 DNA sequencing and sequence analysis of the Tcel DNA polymerase 19
• 1.2 Comparison of the amino acid sequence of the Tcel DNA polymerase 19
• 2. Expression and purification of Tcel DNA polymerase 23
• 3. Properties of Tcel DNA polymerase 25
• 4. Exonuclease activity of Tcel DNA polymerase 27
• 5. Application to PCR amplification 29
• 5.1 Optimal condition of PCR using Tcel DNA polymerase 29
• 5.2 Long-range PCR of Tcel and Tcel plus DNA polymerases 31
• 5.3 Application of Tcel and Tcel plus DNA polymerases in time-saving PCR 34
• 6. Mutant Tcel DNA polymerases 36
• 7. Thermostability of mutant Tcel DNA polymerases 37
• 8. Assay for PCR efficiency 39
• 8.1 Optimization of PCR amplification using mutant DNA polymerases 39
• 8.2 Time-saving PCR by wild-type and mutant DNA polymerases 41
• 8.3 Long-range PCR by wild-type and mutant DNA polymerases 41
• 9. Assay for fidelity 44
• Ⅳ. References 46
• ABSTRACT 51