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

Authors : Hamid CEYLAN, Orhan ERDOĞAN

Pages : 77-87

View : 10 | Download : 6

Publication Date : 2017-12-01

Article Type : Research Paper

Abstract :The molecular structure of acetylcholinesterase insert ignore into journalissuearticles values(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 insert ignore into journalissuearticles values(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 insert ignore into journalissuearticles values(DE-3);. In this work, we produced hexahistidine insert ignore into journalissuearticles values(6xHis); tagged fusion protein by isopropyl β-D-1-thiogalactopyranoside insert ignore into journalissuearticles values(IPTG); induction and purified using nickel insert ignore into journalissuearticles values(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 9.0 glycine-NaOH buffer and room temperature. Kinetic parameters, KM and Vmax, were determined as 0.63 and 0.69, respectively.
Keywords : Acetylcholinesterase, cloning, recombinant protein, enzyme characterization