tailieunhanh - Cloning, expression, and characterization of human brain acetylcholinesterase in Escherichia coli using a SUMO fusion tag

The molecular structure of acetylcholinesterase (AChE) attracts interest because of its versatility and significant role in the cholinergic system. The main purpose of the present study was to clone a full-length cDNA sequence of human brain acetylcholinesterase (hAChE) into pET SUMO vector and express it successfully. | Turkish Journal of Biology Research Article Turk J Biol (2017) 41: 77-87 © TÜBİTAK doi: Cloning, expression, and characterization of human brain acetylcholinesterase in Escherichia coli using a SUMO fusion tag Hamid CEYLAN, Orhan ERDOĞAN* Department of Molecular Biology and Genetics, Faculty of Science, Atatürk University, Erzurum, Turkey Received: Accepted/Published Online: Final Version: Abstract: The molecular structure of acetylcholinesterase (AChE) attracts interest because of its versatility and significant role in the cholinergic system. The main purpose of the present study was to clone a full-length cDNA sequence of human brain acetylcholinesterase (hAChE) into pET SUMO vector and express it successfully. The integrity of the constructed plasmid was confirmed by cross PCR. This recombinant construct was expressed in Escherichia coli BL21 (DE-3). In this work, we produced hexahistidine (6xHis) tagged fusion protein by isopropyl β-D-1-thiogalactopyranoside (IPTG) induction and purified using nickel (Ni2+) affinity chromatography. Using anti-His antibody, we detected ~90 kDa fusion protein. The expression of the hAChE gene in a microbial host resulted in good biological activity. Using the Ellman method, the recombinant AChE exhibited activity with optima at pH glycine–NaOH buffer and room temperature. Kinetic parameters, KM and Vmax, were determined as and , respectively. Key words: Acetylcholinesterase, cloning, recombinant protein, enzyme characterization 1. Introduction Characteristic of cholinergic synapses is termination of synaptic transmission by neurotransmitter hydrolysis (Zimmerman and Soreq, 2006). Acetylcholinesterase (AChE) is present at all cholinergic synapses and exerts an essential role in cholinergic transmission by swiftly hydrolyzing the acetylcholine that is a key component of cholinergic signaling. This function of enzyme .