Coexpression Of PpPDI Effects Secretion Of Bombyx Mori Acetylcholinesterase In Pichia Pastoris

The enzyme inhibition method is an useful method for rapid detection of pesticide residues. Acetylcholinesterase(ACh E) is an important enzyme used for this method to detect Organophosphate pesticide and Carbamate pesticide. At the present stage, acetylcholinesterase is mostly purified from insect and animal serum, but the process is inefficient and the purification process is complex. So it's meaningful and urgent to develop an efficient and cheap method to get the enzyme. In our laboratory's previous work, Bombyx mori acetylcholinesterase(Bm ACh E) gene has been already cloned to p PIC9 K and the recombinant strain GS115/p PIC9K-bmace-opt have been constructed successfully. The expression product recombinant Bm ACh E shows high sensitivity to both Organophosphate pesticide and Carbamate pesticide. But the production is still expected to be enhanced. The strategy, co-expression with protein disulfide isomerase( PDI), is adopted in this study to improve the production of recombinant Bm ACh E successfully. The main results are as follows:(1) Construction of recombinant strain p PIC9K-bmace-opt/PDI The PDI gene fragment was cloned from Pichia pastoris with a size of 1554 bp and sequenced. The sequencing results were alignmented with EMBL(AJ302014) data and the result showed that they have 100% homology. Subsequently, the Asu II enzyme digestion site and the Flag-tag gene fragment were introduced to the 5' end of the PDI gene fragment by PCR amplification, simultaneously, the Xba?enzyme digestion site was introduced to the 3' end. The PCR production of PDI and the empty vector p PICZ?A were digested with the enzyme Asu II and Xba I. After that, the enzyme digestion products were connected and transformed to Escherichia coli TOP10 f '. The p PICZ?A-PDI expression vector was identified to be constructed successfully by Zeocin resistance screening, colony PCR and sequencing. The right p PICZ?A-PDI expression vector was then transformed into therecombinant enzyme strain GS115/p PIC9K-bmace-opt, and it can be integrated to the genome by homologous recombination. The recombinant enzyme strains GS115/ p PIC9K-bmace-opt/PDI was identified by Zeocin resistance selection and colony PCR.(2) Secretory expression of recombinant Bm ACh E The recombinant enzyme strains GS115/p PIC9K-bmace-opt/PDI was induced to express recombinant protein. Via the PAGE-SDS protein gelelectrophoresis and Western blotting of the introcellular protein and extracellular protein respectively, the protein disulfide isomerase was proved to be expressed correctly in the cell and the recombinant Bm ACh E was certified to be expressed and secreted to the supernatant. By analysis of the enzyme activity and the content of crude protein of the both strains within 6 days using Ellman's method and Bradford method, the best enzyme activity and crude protein concentration of expression from GS115/p PIC9K-bmace-opt/PDI and GS115/p PIC9K-bmace-opt were 107.02 U / m L(297 mg / L) and 44.40 U / m L(177 mg / L) respectively. The production of the former is about 2.4 fold of that of the later.(3) Optimization of the secretion expression conditions of recombinant Bm ACh E The expression conditions of recombinant strain were optimized to improve recombinant Bm ACh E. The optimal induction temperature, bacterial density, methanol concentration, p H value were 20 ?, OD600=20, 1%, p H=8.0 respectively, Under the optimal inducing condition, enzyme activity could reach 503 U/m L, and the crude protein concentration could reach 590 mg/L in the 12 th day.(4) Analysis of Bm ACh E activity 7 kinds of representative organic phosphorus and 5 kinds of representative carbamate pesticides were used to analyze Bm ACh E's pesticide sensitivity. The results showed that IC50 of 7 kinds of organic phosphorus pesticides were malathion(5.27×10-6mol/L), methamidophos(6.45×10-6mol/L), trichlorfon(5.53×10-8mol/L), dichlorvos(3.07×10-8mol/L), chlorpyrifos(3.87×10-8mol/L), parathion(2.8×10-8mol/L) and phoxim(3.47×10-9mol/L) respectively. IC50 of 5 kinds of carbamate pesticides were methomyl(9.99×10-8mol/L), aldicarb and Wei(1.1×10-7mol/L), carbaryl(2.45×10-8mol/L), carbofuran(1.91×10-8mol/L), and three hydroxyl carbofuran(1.04×10-8mol/L) respectively. The result was close to the previous pesticides sensitivity data and showed thatcoexpression with PDI has no effect on the pesticides sensitivity of Bm AChE.