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Enhanced Biomass Production of Recombinant Pfu DNA Polymerase Producer Escherichia coli BL21(DE3) by Optimization of Induction Variables Using Response Surface Methodology

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Abstract

Pfu DNA polymerase is one of the most preferred molecular enzymes that is isolated from the hyperthermophilic Pyrococcus furiosus and used for high-throughput DNA synthesis by the polymerase chain reaction. Therefore, an efficient Pfu DNA polymerase production method is necessary for molecular techniques. In the present study, Pfu DNA polymerase was expressed in recombinant Escherichia coli BL21(DE3) and significant parameters for the biomass production were optimized using the central composite design which is the most popular method of response surface methodology. Induction conditions including cell density prior induction (OD600nm), post-induction temperature, IPTG concentration, and post-induction time and their interactions on biomass production were investigated. The maximum biomass production (14.1 g/L) in shake flasks was achieved using the following predicted optimal conditions: OD600nm before induction of 0.4 and the induction at 32 °C for 7.7 h, with 0.6 mM IPTG. Optimized culture conditions were implemented to scale up experiments. 22% and 70% increase in biomass production was achieved in 3 L and 10 L bioreactors, respectively as compared to initial biomass production observed in unoptimized conditions. Similary, a 30% increase of Pfu DNA polymerase production was obtained after the optimization. The polymerase activity of the purifed Pfu DNA polymerase was assessed by PCR amplification and determined as 2.9 U/μl by comparison with commercial Pfu DNA polymerase. The findings of this study indicated that the proposed fermentation conditions will contribute to further scale‑up studies to enhance the biomass for the production of other recombinant proteins.

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All data generated or analyzed during this study are included in this published article.

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Acknowledgements

This work is supported by the Scientific Research Project Fund of Tokat Gaziosmanpaşa University under project number 2019/39. The author is grateful to Professor Bernard A. Connolly (University of Newcastle, UK) and Professor İsa Gökçe (Tokat Gaziosmanpaşa University, Turkey) for supplying The pET-17b(pfu-Pol) recombinant plasmid. The author thanks Professor Murat Elibol (Ege University, Turkey) for assistance with response surface methodology, Research Assistant Ahmet Düzel and Master of Science Cafer Meydan (Ege University, Turkey) for assistance with the bioreactor experiments, Dr. Rizvan İmamoğlu (Bartın University, Turkey) for supplying the strain E. coli BL21(DE3), Professor Muhammed Bahattin Tanyolaç for providing the necessary laboratory equipment for the activity experiments.

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  1. Department of Basic Pharmaceutical Sciences, Faculty of Pharmacy, Tokat Gaziosmanpaşa University, 60250, Tokat, Turkey

    Hülya Kuduğ Ceylan

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HKC performed the experiments, analyzed the data, wrote the main manuscript, prepared its figures and revised the manuscript. reviewed the manuscript.

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Ceylan, H.K. Enhanced Biomass Production of Recombinant Pfu DNA Polymerase Producer Escherichia coli BL21(DE3) by Optimization of Induction Variables Using Response Surface Methodology. Protein J 42, 451–462 (2023). https://doi.org/10.1007/s10930-023-10122-8

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