Cloning, Expression, And Characterization Of Recombinant Mouse Brain Acetylcholinesterase

Acetylcholinesterase (AChE) is an enzyme that hydrolyzes the neurotransmitter acetylcholine into choline and an acetate group at the synaptic cleft. Thus, it has an important role in the transmission of nerve impulses of the parasympathetic nervous system. Certain inhibitors such as Alzheimer’s drugs, organophosphates, and insecticides can block the function of AChE leading to an accumulation of acetylcholine. The excess acetylcholine,(ACh, the impulse-transmitting substance in the synaptic cleft) can cause neuromuscular paralysis throughout the body which can lead to death by asphyxiation.Because of the vital role that AChE plays in proper functioning of the body, it can be beneficial to study AChE effects in vivo through its cloning and expression. Knowing its role in proper signal transduction, many pesticides are used to target the AChE enzyme and inhibit its function. Since traditional pesticide detection methods call for directly extracting AChE from mammalian organs or blood, the purification of AChE using this method has many disadvantages such as a low output, high cost, instable sensitivity and the use of complex instrument structures. Due to these circumstances, the use of molecular cloning techniques and recombinant protein expression along with purification techniques are useful methods to produce AChE in large amounts and high purity.For this study, the cDNA of AChE taken from the Kunming mouse was cloned by reverse transcription polymerase chain reaction (RT-PCR). The recombinant plasmid pMEK-ace was constructed by subcloning the AChE gene into pMEKml2, and it was expressed at high levels in E. coli BL21competent cells. The specific activity of the purified protein reached742U/mg after purified2,400fold. The maximum activity of the recombinant enzyme reacting with the substrate ATChI was at40℃and7.5pH. These results provide useful information showing how various conditions have a direct effect on acetylcholinesterase suggesting it to be an important key target of pesticides.