Preparation Of High-purity Recombinant Bombyx Mori Type ? Acetylcholinesterase

The enzyme inhibition method is still the mainstream method for the rapid detection of pesticides.In our previous work,we found that rBmAChE2 showed good sensitivity to organophosphorus pesticides and carbamate pesticides,suggesting that it may be used as a new enzyme source for rapid detection of pesticides.In order to further understand the crystal structure characteristics of the enzyme for the later molecular evolution,it is necessary to prepare a high-purity enzyme protein.For this reason,based on the analysis of the biological information of the nucleic acid and protein sequence of the BmAChE2,E.coli expression system and Pichia pastoris expression system were selected,respectively,to prepare high-purity enzyme proteins by trying different expression strategies,optimizing the separation and purification methods,which will lay the foundation for the later theoretical research of enzyme proteins.The main research contents and results are as follows:?1?Bioinformatics analysis of BmAChE2Using NCBI,PDB,SignalP 4.1 Server,Transmembrane Prediction server,ExPASy Proteomics tools,Phyre2 and other databases and online servers and related software,The BmAChE2 was analyzed in detail from five aspects:nucleic acid sequence analysis,primary structure analysis and partial physicochemical property prediction,secondary structure prediction,tertiary structure and homology comparison.Bmace2 is 1917 bp in size and encodes 638 amino acids.The molecular formula of BmAChE2 was C₃₂₅₄H₄₉₁₂N₈₃₀O₉₃₇S₃₁,the molecular weigh was 71645.66Da.It contains 48 E.coli rare codons:GGA 21,ATA 7,AGA 5,CTA 4,CCC 4,AGG2,CGA 2,CGG 2,TCG 1.The theoretical isoelectric point was 5.49.There were 70acidic amino acid residues and alkaline amino acid residues respectively,accounting for 10.97%.There was a signal peptide consisting of 23 amino acids at the N-terminus of BmAChE2 and a hydrophobic tail consisting of 17 amino acids at the C-terminus.The percentage of for each secondary structure was calculated.Among them,the random coil content was the highest,reaching 50%,alpha helix accounting for 36%,beta sheet accounting for 11%,TM helix accounting for 3%.The TM helix was just at the C-terminus,in accordance with the“DAS”predicted,and other parts of C-terminal hydrophobic tail were composed of random coil.Besides,BmAChE2 had9 Cys,which may form four pairs of intramolecular disulfide bonds and one intermolecular disulfide bond.There were also three potential N-glycosylation sites,namely N138,N321,and N522,they all located on the surface of the BmAChE2structure and were away from the active gorge.The homologous alignment of the amino acid sequence of BmAChE2 with other sources of AChE published in the PDB was conducted.Among them,BmAChE2 and Drosophila AChE had the highest homology,up to 50%.The N-terminal signal peptide and C-terminal hydrophobic tail of BmAChE2 were not present at the ends of the other crystallized AChE.Maybe these two parts were not necessary for the maintenance of the AChE tertiary structure.?2?Expression of BmAChE2 in E.coliBased on the results of bioinformatics analysis of BmAChE2,the rare codons,disulfide bond formation,and protein solubility were considered as the the major influencing factors of BmAChE2 expression in E.coli to explore the role of different expression strategies.The use of Transetta?DE3?reduced the impact of rare codons on expression;the removing of the signal peptide and hydrophobic peptide from the BmAChE2 fragment,the using of PelB signal peptide and the fusion expression with MBP/GSTtag promoted the soluble expression of the target protein;the co-expression with PDI and the use of E.coli Origami2 as the host promoted the formation of the disulfide bond of the target protein.Finally,four expression vectors?pGEX-4T-1-Bmace2,pET22b-Bmace2,pET30b-Bmace2/pBAD-myc-hisA-PDI and pMAL-P2E-Bmace2?were constructed,and three expression host strains were selected?BL21?DE3?,Transetta?DE3?,Origami2?.The results showed that the target protein was mainly expressed in the form of inclusion body in E.coli.When the vector and host were combined as pGEX-4T-1/Origami2,there was a little soluble protein in the cell,and the yield of purified protein was low when purified by Ni column.Thus the expression conditions of inclusion bodies was optimized,and the expression level was highest when the concentration of IPTG was 0.3 mM and the temperature was 18°C for 30h.After refolding and denaturation,the purity of BmAChE2 reached more than 90%,but there was no activity of acetylcholinesterase.?3?The expression of BmAChE2/M₂₃ in Pichia pastorisThe Pichia pastoris engineered strain pPIC9K-ace-opt-GS115?which had been codon-optimized?obtained in the laboratory was induced to express BmAChE2,and the purification conditions were optimized.After three-step purification with Ni column,Q column and SEC column,the enzyme activity was determined to be 701.57U/mL and the specific activity was 2300 U/mg,but the SDS-PAGE results showed that the target protein had severe tailing.After treatment with PNGase F,the tail disappeared,suggesting that the N-glycosylation modification caused tailing.Based on the predicted N-glycosylation sites,N321 and N522 were mutated to construct the mutant recombinant vector pPIC9K-ace-opt-M23?N321Q,N522Q?,which was transferred into Pichia pastoris GS115 genome and the recombinant mutant strain pPIC9K-ace-opt-M23-GS115 was obtained.After induced by methanol,the supernatant was taken and purified by Ni column.The activity of BmAChE2/M₂₃was determined.The specific activity of BmAChE2/M23 was found to be 1137.33U/mg,indicating that BmAChE2/M₂₃ retained acetylcholinesterase activity after N321Q and N522Q glycosylation mutations.At the same time,the SDS-PAGE analysis found that the tailing phenomenon of BmAChE2 disappeared after the mutation,and the purity of the target protein was above 90%,which met the requirement for crystal protein purity.