A simple method for in-house Pfu DNA polymerase purification for high-fidelity PCR amplification
Background and Objectives: Pfu DNA polymerase is an enzyme that exhibits the lowest error rate in the 3´ to 5´ exonuclease (proofreading) activity during DNA synthesis in Polymerase Chain Reactions (PCRs). This study was aimed to express and purify Pfu DNA polymerase in a bacterial expression system...
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| Format: | Article |
| Language: | English |
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Tehran University of Medical Sciences
2019-05-01
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| Series: | Iranian Journal of Microbiology |
| Subjects: | |
| Online Access: | https://ijm.tums.ac.ir/index.php/ijm/article/view/1929 |
| _version_ | 1818876208795877376 |
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| author | Prabu Siva Sankar Marimuthu Citartan Aminah Ahmed Siti Boris V. Skryabin Timofey S. Rozhdestvensky Goot Heah Khor Thean Hock Tang |
| author_facet | Prabu Siva Sankar Marimuthu Citartan Aminah Ahmed Siti Boris V. Skryabin Timofey S. Rozhdestvensky Goot Heah Khor Thean Hock Tang |
| author_sort | Prabu Siva Sankar |
| collection | DOAJ |
| description | Background and Objectives: Pfu DNA polymerase is an enzyme that exhibits the lowest error rate in the 3´ to 5´ exonuclease (proofreading) activity during DNA synthesis in Polymerase Chain Reactions (PCRs). This study was aimed to express and purify Pfu DNA polymerase in a bacterial expression system under a simple purification method.
Materials and Methods: Pfu polymerase gene sequence, derived from Pyrocuccus furiosus (Pfu) genomic DNA, was cloned and overexpressed in E. coli BL21 (DE3) pLysS. Upon overexpression, bacterial lysate containing the Pfu DNA polymerase was heated at 94°C for 5 minutes. Pfu DNA polymerase having high thermal stability was retained while the other bacterial proteins were denatured. The resulting thermo stable Pfu DNA polymerase was separated from the other debris of the denatured proteins by simple centrifugation.
Results: The enzymatic activity of the resulting Pfu DNA polymerase was estimated by comparing with the commercial Pfu DNA Polymerases. An estimated 50000 units of functional Pfu DNA polymerase was produced from a 400 ml culture.
Conclusion: The in-house produced Pfu DNA Polymerase could be used for routine amplification that requires high-fidelity such as cloning and DNA sequencing. |
| first_indexed | 2024-12-19T13:38:45Z |
| format | Article |
| id | doaj.art-8c0f6334da53419cbf36a747f1cc7fec |
| institution | Directory Open Access Journal |
| issn | 2008-3289 2008-4447 |
| language | English |
| last_indexed | 2024-12-19T13:38:45Z |
| publishDate | 2019-05-01 |
| publisher | Tehran University of Medical Sciences |
| record_format | Article |
| series | Iranian Journal of Microbiology |
| spelling | doaj.art-8c0f6334da53419cbf36a747f1cc7fec2022-12-21T20:19:05ZengTehran University of Medical SciencesIranian Journal of Microbiology2008-32892008-44472019-05-0111210.18502/ijm.v11i2.1085A simple method for in-house Pfu DNA polymerase purification for high-fidelity PCR amplificationPrabu Siva Sankar0Marimuthu Citartan1Aminah Ahmed Siti2Boris V. Skryabin3Timofey S. Rozhdestvensky4Goot Heah Khor5Thean Hock Tang6Advanced Medical & Dental Institute (AMDI), Universiti Sains Malaysia, Bertam, Kepala Batas, Penang, MalaysiaAdvanced Medical & Dental Institute (AMDI), Universiti Sains Malaysia, Bertam, Kepala Batas, Penang, MalaysiaAdvanced Medical & Dental Institute (AMDI), Universiti Sains Malaysia, Bertam, Kepala Batas, Penang, MalaysiaFaculty of Medicine (TRAM), University of Muenster, Muenster, GermanyFaculty of Medicine (TRAM), University of Muenster, Muenster, GermanyCentre of Preclinical Science Studies, Faculty of Dentistry, Universiti Teknologi MARA, Sungai Buluh Campus, Selangor, MalaysiaAdvanced Medical & Dental Institute (AMDI), Universiti Sains Malaysia, Bertam, Kepala Batas, Penang, MalaysiaBackground and Objectives: Pfu DNA polymerase is an enzyme that exhibits the lowest error rate in the 3´ to 5´ exonuclease (proofreading) activity during DNA synthesis in Polymerase Chain Reactions (PCRs). This study was aimed to express and purify Pfu DNA polymerase in a bacterial expression system under a simple purification method. Materials and Methods: Pfu polymerase gene sequence, derived from Pyrocuccus furiosus (Pfu) genomic DNA, was cloned and overexpressed in E. coli BL21 (DE3) pLysS. Upon overexpression, bacterial lysate containing the Pfu DNA polymerase was heated at 94°C for 5 minutes. Pfu DNA polymerase having high thermal stability was retained while the other bacterial proteins were denatured. The resulting thermo stable Pfu DNA polymerase was separated from the other debris of the denatured proteins by simple centrifugation. Results: The enzymatic activity of the resulting Pfu DNA polymerase was estimated by comparing with the commercial Pfu DNA Polymerases. An estimated 50000 units of functional Pfu DNA polymerase was produced from a 400 ml culture. Conclusion: The in-house produced Pfu DNA Polymerase could be used for routine amplification that requires high-fidelity such as cloning and DNA sequencing.https://ijm.tums.ac.ir/index.php/ijm/article/view/1929Protein expressionPyrococcus furiosusDNA polymerasePolymerase chain reaction |
| spellingShingle | Prabu Siva Sankar Marimuthu Citartan Aminah Ahmed Siti Boris V. Skryabin Timofey S. Rozhdestvensky Goot Heah Khor Thean Hock Tang A simple method for in-house Pfu DNA polymerase purification for high-fidelity PCR amplification Iranian Journal of Microbiology Protein expression Pyrococcus furiosus DNA polymerase Polymerase chain reaction |
| title | A simple method for in-house Pfu DNA polymerase purification for high-fidelity PCR amplification |
| title_full | A simple method for in-house Pfu DNA polymerase purification for high-fidelity PCR amplification |
| title_fullStr | A simple method for in-house Pfu DNA polymerase purification for high-fidelity PCR amplification |
| title_full_unstemmed | A simple method for in-house Pfu DNA polymerase purification for high-fidelity PCR amplification |
| title_short | A simple method for in-house Pfu DNA polymerase purification for high-fidelity PCR amplification |
| title_sort | simple method for in house pfu dna polymerase purification for high fidelity pcr amplification |
| topic | Protein expression Pyrococcus furiosus DNA polymerase Polymerase chain reaction |
| url | https://ijm.tums.ac.ir/index.php/ijm/article/view/1929 |
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