Expression And Purification Of Recombinant Human Acetylcholinesterase And Screening Of Its Inhibitors

Acetylcholinesterase, as a kind of biological catalyst, which is composed of protein molecules in the synaptic cleft of the cholinergic synapse, is a key enzyme in the process of biological nerve conduction. It is not only involved in the growth and development of cells, but also on the regeneration of nerve cells and the development of neurons. It plays a very important role in maintaining the normal physiological function of the human nervous system. Acetylcholine is the substrate of AChE, ACh can be affected by the catalytic reaction of AChE, resulting in the absence of ACh, which makes the nerve signal transmission failure.And the decrease of ACh level or the abnormal activity is the main reason for the occurrence of Alzheimer’s disease. Therefore, inhibiting AChE to restore or improve the level of ACh is an effective method to treat AD. AChE inhibitors can selectively inhibit the activity of AChE in the brain; also it can prolong the survival time in the brain and improve the function of the AD patients.So far, AChEI has become the mainstream product for the treatment of mild and moderate AD. The approved listings of the six AChEI by FDA are tacrine, galantamine, Huperzia A, new compound Namzari and donepezil. But they have been found that the AChEI have many disadvantages, such as short half-life, toxicity and serious side effect of severe cholinergic system, which makes them in the clinical application of the above subject to certain restrictions. Therefore, a new generation of AChEI is still a hot spot in the treatment of AD.The aim of this study was to establish the expression system of recombinant human acetylcholinesterase using human embryonic kidney cells in vitro. pCMV-AChE plasmid was used to carry out the transient transfection, and the expression of the enzyme was secreted into the culture medium. The activity of the recombinant protein was investigated by the study of enzyme kinetics. FF DEAE-Sepharose affinity chromatography was used to purify the recombinant proteins, and to obtain the high purity and activity of recombinant human acetylcholinesterase,so as to establish a screening model for the evaluation of recombinant human acetylcholinesterase inhibitors, and to determine the inhibitory activity of Chinese herbal medicine and its derivatives from natural products and their derivatives.1, HEK293 cells secrete recombinant human acetylcholinesteraseThe pCMV-AChE plasmid was donated by the College of medicine, Zhejiang University. The DNA sequence of pCMV-AChE was determined by AChE sequence and the sequence was consistent with AChE gene. HEK293 cells were transiently transfected with pCMV-AChE by cationic liposome lipofectamineTM 2000. It secretions of rhAChE into the cell culture medium. Cell culture fluid was collected and AChE activity was analyzed. Finally, the crude enzyme solution was obtained by the next step.2, rhAChE activity and detection systemUsing Ellman’s method of enzymatic activity assay: reaction system with enzyme solution and the chromogenic agent DTNB, pH 7.4 PBS, at 37℃ culture incubator 6min, after the addition of substrate acetylthiocholine iodide 37℃ to incubation 16 min, and finally adding SDS termination reaction. Enzyme labeling instrument is used to measure at 412 nm absorbance(OD). rhAChE reaction rate unit uses OD/min to represent.3, Purification and identification of rhAChEIn order to obtain higher purity and higher activity of rhAChE, crude enzyme liquid need to be purified. At first, after the activity of the enzyme was analyzed, using dialysis bags make it for all night. The dialysis fluid was purified by FF DEAE-Sepharose affinity chromatography column gradient elution.Collecting protein purification, detecting its activity.Using polyethylene glycol concentrates 5 times. The concentration of BCA was 0.145mg/mL, the purification efficiency was 9.63, and the recovery of enzyme activity was 56%. The relative molecular mass of the enzyme is about 64 KD. To acetylthiocholine iodide as substrate, the optimum reaction temperature is 37℃ of the enzyme; the optimum pH was 7.4 of the enzyme. The optimum substrate concentration is 5mmol/L of the enzyme. Using SDS-PAGE and Native-PAGE identification, there was a single band near the molecular weight of 64 KD. Western-Blot analysis of purified liquid, the frist antibody uses Anti-AChE antibody, there was a single rhAChE band near the molecular weight of 64 KD.Because the purity of recombinant human acetylcholinesterase can directly determine the accuracy and reliability of the final detection.This experiment has done a research on the separation and purification of human acetylcholinesterase and the biological significance of this experiment. That is, how to achieve the maximum, reduce and simplify the purification efficiency of rhAChE, and reduce the loss of the enzyme activity to improve the purification efficiency.4, Screening of rhAChE inhibitorsDonepezil is reversible inhibitor of AChE.The IC50 of crude enzyme is 14.0 nmol/L. This study selected 7 donepezil concentration to determine the effect of inhibition rate of purified liquid rhAChE. The negative logarithm of the concentration of the compound and the inhibition rate of the enzyme came to linear regression.Get the IC50 of rhAChE. Reaction conditions: pH was 7.4.The amount of enzyme was 5 μL, The concentrations of donepezil: 0, 5.0, 10.0, 20.0, 40.0, 80.0, 160 nmol/L, substrate concentration: 0.5mmol/L, reaction temperature of 37℃, reaction time is 16 min. The IC50 of rhAChE was significantly decreased to about 5.0 nmol/L after the test.Using the above model of human acetylcholinesterase inhibitor to inhibit the activity of rhAChE and the screening of compounds. Determination and identification of effective parts of evaluation has significant inhibitory activity. It can be more efficient and quick to screen and identify the components of acetylcholinesterase inhibitors from natural drugs.