Expression Of Drosophila Melanogaster Acetylcholinesterase (AChE) Gene In Pichia Pastoris

Pesticide makes a great contribution to agriculture in preventing and curing insect pest,but the widely spreadable and unreasonable use of pesticide causes environmental pollution. It is harmful to human and other life-form. So it is necessary and important to detect rudimental pesticide in environment and food. Traditional methods used for the detection of insecticide are based on gas chromatography (GC) or high performance liquid chromatography (HPLC), which are expensive and sophisticated analytical techniques, and not suitable for the situation of China. So we need an easier, faster and cheaper method to detect the pollution of pesticides.Acetylcholinesterase (AChE, EC 3.1.1.7) is hydrolase existing in central nerve system and plays a key role in cholinergic transmission by hydrolysing the neurotransmitter acetylcholine into acetate and choline. Organophosphate and carbamate pesticide kills pest by inhibiting cholinesterase in central nerve of insects. As the most optimum substrate of organophosphate and carbamate pesticide, AChE was widely used in detection of rudimental pesticide. Especially AChE-based biosensor has been focus in developing various detection technologies.At present, AChEs are mainly extracted from insect or animal blood. The disadvantages of this method are low yield, high cost and unstable sensitivity, which limit the use of AChEs in detecting rudimental pesticide. Along with development of gene engineering technology, We can resolve this problem by cloning sensitive AChE genes and efficiently expressing them in heterologous expression systems. DmAChE is more sensitive than other AChEs, and its cDNA sequence has been known. As a eukaryota, Pichia pastoris is a efficient eukaryotic expression systems which has many advantages such as protein processing, protein folding, posttranslational modification and convenient and economical culture, and so on. It is faster, easier, and lower cost than other eukaryotic expression systems, and we can generally obtain higher yield of expression products. So we constructed expression vector pPIC9K/DmAChE and expressed the DmAChE gene in P.pastoris.We cloned the cDNA encoding AChE gene of Drosophila melanogaster (DmAChE1) by PCR with primer Pp1 and Pp2 and cloned the cDNA of DmAChE without GPI encoding sequence (DmAChE2) with primer DmAC1 and DmAC2. The cDNA encoding DmAChE1 and DmAChE2 were respectively inserted into pPIC9K. The recombinant plasmids of pPIC9K-DmAChE1 and pPIC9K-DmAChE2 were successfully transformed into Pichia pastoris KM71 by electrotransformation. In shake-flask culture induced by methonal, the AChE activity in supernatant was detected by improved Ellman method. The result showed that DmAChE2 was efficiently expressed and secret into culture medium, but DmAChE1can't. The number of copy of DmAChE gene has no relation to DmAChE expression yield. Optimum culture medium and culture conditions have been optimized. Isolate DmAChE2-331 with the highest expression activity was screened out and the yield of DmAChE which the isolate expressed achieved 37.5U/ml at 168 hours after methanol induction. Through purification using metal-chelate affinity chromatography, We found that there were two processing forms of recombinant DmAChE in Pichia pastoris.