A simple method for in-house Pfu DNA polymerase purification for high-fidelity PCR amplification
| Autor: | Aminah Ahmed Siti, Thean-Hock Tang, Prabu Siva Sankar, Goot Heah Khor, Boris V. Skryabin, Timofey S. Rozhdestvensky, Marimuthu Citartan |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Microbiology (medical)
Exonuclease DNA polymerase viruses lcsh:QR1-502 complex mixtures 01 natural sciences Microbiology lcsh:Microbiology DNA sequencing law.invention law 0502 economics and business 0101 mathematics Polymerase Polymerase chain reaction Pfu DNA polymerase DNA synthesis biology Chemistry 010102 general mathematics 05 social sciences virus diseases biochemical phenomena metabolism and nutrition Molecular biology Pyrococcus furiosus genomic DNA biology.protein Protein expression 050203 business & management |
| Zdroj: | Iranian Journal of Microbiology, Vol 11, Iss 2 (2019) |
| ISSN: | 2008-4447 2008-3289 |
| Popis: | Background and Objectives: Pfu DNA polymerase is an enzyme that exhibits the lowest error rate in the 3´ to 5´ exonucle- ase (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 dena- tured proteins by simple centrifugation. Results: The enzymatic activity of the resulting Pfu DNA polymerase was estimated by comparing with the commer- cial 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|