University of Isfahan Journal of Microbial Biology 3060-7647 8 32 2019 12 22 Cloning and Expression of Bst DNA Polymerase I Gene in E. coli BL21‎ Cloning and Expression of Bst DNA Polymerase I Gene in E. coli BL21‎ 41 46 22964 10.22108/bjm.2018.110084.1116 FA Zahra Emami Biotechnology Dept, Faculty of Biological Sciences, Alzahra University, Tehran, Iran Sara Gharavi Biotechnology Dept., Faculty of Biological Sciences, Alzahra University, Tehran, Iran Ezat Asgarani Biotechnology Dept faculty of Biological Sciences Alzahra University Tehran Iran Journal Article 2018 03 13 Introduction: DNA polymerases, in addition to being indispensable in replication and repair, are also very useful in a number of molecular biology techniques such as DNA amplification, site-directed mutagenesis, DNA sequencing, different kinds of PCR, loop-mediated isothermal amplification (LAMP), etc. After the invention of PCR, efforts have been made to focus on the identification and isolation of thermo-tolerant enzymes that amplify DNA efficiently at high temperatures. Materials and methods: In this study, Geobacillus stearothermophilus strain10 was selected for the cloning of Bst DNA Pol I - encoding gene. Following DNA extraction from the bacterium, PCR was carried out to amplify the pol A gene using designed primers and to clone via pET32a expression vector followed by transfer to the heterologous E.coli BL21 host. The cloned gene was expressed by induction with IPTG and the resultant protein purified by IMAC column. Results: The activity of the functional fragment was assessed by LAMP and showed a relatively high DNA amplification ability in comparison with commercial Bst DNA Polymerase which is usually used in this amplification protocol. Discussion and conclusion: This study found thatKlenow fragment of recombinant Bst DNA Pol I can amplify uidA gene in E. coli ATCC25923 during the LAMP reaction. Separation of two fragments of the enzyme can improve the activity of Klenow fragment of enzyme in LAMP. Introduction: DNA polymerases, in addition to being indispensable in replication and repair, are also very useful in a number of molecular biology techniques such as DNA amplification, site-directed mutagenesis, DNA sequencing, different kinds of PCR, loop-mediated isothermal amplification (LAMP), etc. After the invention of PCR, efforts have been made to focus on the identification and isolation of thermo-tolerant enzymes that amplify DNA efficiently at high temperatures. Materials and methods: In this study, Geobacillus stearothermophilus strain10 was selected for the cloning of Bst DNA Pol I - encoding gene. Following DNA extraction from the bacterium, PCR was carried out to amplify the pol A gene using designed primers and to clone via pET32a expression vector followed by transfer to the heterologous E.coli BL21 host. The cloned gene was expressed by induction with IPTG and the resultant protein purified by IMAC column. Results: The activity of the functional fragment was assessed by LAMP and showed a relatively high DNA amplification ability in comparison with commercial Bst DNA Polymerase which is usually used in this amplification protocol. Discussion and conclusion: This study found thatKlenow fragment of recombinant Bst DNA Pol I can amplify uidA gene in E. coli ATCC25923 during the LAMP reaction. Separation of two fragments of the enzyme can improve the activity of Klenow fragment of enzyme in LAMP.

https://bjm.ui.ac.ir/article_22964_50dac033c6c5c3501cf961aea2e677fc.pdf