SF-GFP-mediated Cell Surface Display Of Methyl Parathion Hydrolase In Escherichia Coli

Methyl parathion Hydrolase is derived from a Pseudomonas sp.M6 which capable of degrading methyl paration.MPH can effectively and specifically degrade methylparathion.Methyl parathion,paraoxon,parathionand other carbon-phosphorus bonds or organic groups containing phosphoric acid derivatives,are the main ingredients of organic phosphorus insecticide,organic phosphorus herbicide,organic phosphorus nerve gas and et al.The toxicity of these components to the organism is mainly manifested in the inhibition of acetylcholinesterase activity,resulting in a large accumulation of acetylcholine on the posterior synaptic membrane,leading to excessive stimulation of the nervous system.Blamed for the widespread use of organophosphorus pesticides in many countries,this situation has led to serious water,soil,atmospheric environmental pollution and ecological damage,a serious threat to human life safety.Although a variety of organophosphorus hydrolases have been found,but the low expression level,high production costs hinder their industrial production.In this study,by using N-terminal of GFP as anchoring protein,a genetically engineered Escherichia coli(E.coli)strain surface displayed Methyl parathion Hydrolase(mph)with improved enzyme activity was successfully constructed,The specific experimental results are as follows:1.Gene synthesis of methyl parathion hydrolase MPH-RThe mph gene from Pseudomonas sp.M6 was modified according to the preference principle of the E.coli codon,and a new methyl parathion hydrolase gene mph-r was redesigned and optimized.Afterwards,according to the designed gene sequence,overlapping PCR primers were designed by DNA woking.The new gene sequence mph-R gene was 930 bp in length and encoded a total of 309 amino acids,of which the C-terminal HA tag.2.Construction and Expression of pET23a-sfgfp-mph Expression PlasmidAfter the target gene was obtained,mph was successfully ligated into pET23a-sfgfp vector by T5 cloning,and the pET23a-sfgfp-mph-r plasmid was successfully constructed.The constructed expression plasmid was rapidly transformed into the competent state of Rosetta blue.The strain with green fluorescence was picked and inoculated in LA liquid medium,and cultured at 37?.until OD600=0.6,and induced at 18? for 24 h by IPTG The bacteria were collected and detected by SDS-PAGE.3.Surface display of sfGFP-MPH fusion proteinThe cultured cells were processed to obtain the outer membrane,periplasmic space,inner membrane and whole cell proteins,and the SDS-PAGE and Western blot were used to identify the localization of MPH in the cells.Since the fusion protein has sf-GFP at the N-terminus and HA tag at the C-terminus,we immunofluorescently labeled the HA tag with a fluorescent antibody.Further experiments showed that the display efficiency of the fusion protein was approximately 40%as detected by flow cytometry.It was observed by fluorescence confocal microscopy that the protein was indeed displayed on the cell surface.It was observed by fluorescence confocal microscopy that the protein was indeed displayed on the cell surface.So far,there are not many reports on the display of MPH using the E.coli display system.Even though there are many reports of ice nuclein,the expression level of MPH in the prokaryotic expression system is very low,so the display efficiency is extremely low.By using sf-GFP as the N-terminal fusion tag of MPH,the expression and display efficiency of MPH in E.coli were greatly improved.4.Characterization of the activity and stability of the surface-immobilizedAfter treatment of the cultured cells,whole cell activity was measured spectrophotometrically by using methyl parathion as a substrate in the reaction mixture.After determination,the optimum temperature for the whole cell was 30?,the optimum pH was 9,and the highest enzymatic activity of the whole cell was 11.4U.Metal ions were used as additives to detect changes in whole cell activity.When the concentration was 1 mM,Co2 + could effectively increase the activity of whole cells reaching 151%,respectively.By standing the harvested cells at room temperature,we found that the cells were still able to maintain a certain level of activity and that they were able to continuously degrade methyl parathion.