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Isolation and recombinant Tth DNA polymerase expression in E. coli BL21, purification and enzyme activity investigation
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Elham Soleimani *   , Camellia Katalani , Behzad Shahin-Kaleybar , Ghorbanali Nematzadeh |
| Sari Agricultural Sciences and Natural Resource University (SANRU) & Genetics & Agricultural Biotechnologhy Institute of Tabarestan (GABIT) , solimanielham@yahoo.com |
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Abstract: (18 Views) |
| The discovery of thermostable DNA polymerase from thermophilic microorganisms has been a significant advancement in PCR-related techniques in molecular biology and genetic engineering. Tth DNA polymerase (Tth pol) from the thermophilic bacterium Thermus thermophilus is extensively used in genetic engineering. Tth pol possesses reverse transcriptase and polymerase activity, tolerance to PCR inhibitors in biological samples, and a higher extension rate compared to Taq DNA polymerase, making it an attractive enzyme for researchers to use. In this study, the gene sequence of Tth pol was isolated and transformed into E.coli DH5α after ligation to pTZ57R/T. The recombinant plasmid sequencing confirmed the correct insertion. Tth pol was then amplified with specific primers and ligated to pET28a. The recombinant plasmid was transformed into E.coli BL21. Expression induction was performed with 0.8 and 1 mM IPTG in LB and modified minimal medium at 25 °C and 30 °C. Samples were taken at 2-hour intervals for up to 10 hours after induction. SDS PAGE of the samples revealed that the optimum condition for Tth pol expression was 30 °C, with 1 mM IPTG in modified minimal medium and 10 hours after induction. Due to the presence of a histidine tag in the recombinant Tth pol enzyme, nickel resin was used for purification. The purified Tth pol activity was assessed in PCR, and its reverse transcription activity was evaluated in cDNA synthesis. The results revealed that this enzyme has both activities. The findings from this study recommend optimizing cultivation conditions on a large scale, such as in a fermentor, to achieve higher expression of Tth pol. |
Article number: 4 |
| Keywords: Tth DNA polymerase, recombinant protein expression, purification, culture medium |
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Full-Text [DOC 1353 kb]
(11 Downloads)
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Type of Study: Research |
Subject:
Microrganisms and Viruses
Received: 2025/01/25 | Accepted: 2025/03/10 | Published: 2025/11/15
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