Cloning and Expression of Rat Brain Acetylcholinesterase Enzyme in Escherichia coli

Year 2022, Volume: 12 Issue: 1, 287 - 296, 01.03.2022

Ahmet Karakuş , Hamid Ceylan , Orhan Erdoğan

https://doi.org/10.21597/jist.962268

Abstract

In this study, the gene region of rat (Rattus norvegicus) brain acetylcholinesterase enzyme was amplified in PCR with designed forward and reverse primers and it was ligated into a pET-SUMO vector under suitable conditions. This recombinant vector was transformed to competent Escherichia coli cells and it was grown in liquid LB medium including kanamycin. Colony PCR was performed from growing colony and PCR products were checked with agarose gel electrophoresis. The correct colonies were grown in a liquid medium for plasmid isolation. After plasmid isolation, these recombinant constructs were used for whether the gene inserts properly with cross-PCR. After determining the accuracy of the plasmid, recombinant vectors were transferred into the E. coli BL21 (DE3) cells to perform protein production. Cells were grown in IPTG induced larger media for hours. Enzyme activity and SDS-PAGE analysis were performed from homogenate for each treatment samples.

Keywords

Cloning, pET-SUMO vector, E. coli, Rattus norvegicus, Acetylcholinesterase

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