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Expression optimization, purification and activity of recombinant Tth DNA polymerase in E. coli BL21
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Elham Soleimani *    , Camellia Katalani , Behzad Shahin-Kaleybar , Ghorbanali Nematzadeh |
| Plant Biotechnology Dept., Genetics & Agricultural Biotechnology Institute of Tabarestan (GABIT) Sari Agricultural Sciences and Natural Resource University, Iran , solimanielham@yahoo.com |
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Abstract: (383 Views) |
The discovery of thermostable DNA polymerase from thermophilic microorganisms has been a significant advancement 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 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. |
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| Keywords: Tth DNA polymerase, recombinant protein expression, purification, culture medium |
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Type of Study: Research |
Subject:
Microrganisms and Viruses
Received: 2025/01/25 | Accepted: 2025/03/10 | Published: 2025/08/15
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