Construction,production, and characterization of recombinant scFv antibodies against methamidophos expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis> |
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Authors: | Tiejun Li Jianren Cheng Baishi Hu Yan Liu Guoliang Qian Fengquan Liu |
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Institution: | (1) Key Lab of Monitoring and Management of Plant Diseases and Pests, Ministry of Agriculture, College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China |
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Abstract: | Pichia pastoris was used to express a recombinant scFv antibody against methamidophos derived from a recombinant phage-display library. The
specific scFv gene was amplified from a positive clone and then subcloned into the expression vector pPICZα C. The resulting
plasmid, pPICZα C–scFv, was linearized and transformed into P. pastoris (X-33). A transformant named X-33-Pp-Met-28D4, which showed strong expression of antibodies, was isolated, and the culture
conditions were optimized. Under optimal conditions, P. pastoris cultures yielded much higher levels of scFv product than the Escherichia coli expression system. Immunochemical characterization of the scFv antibodies produced in P. pastoris indicated that the affinity and specificity of scFv against methamidophos are comparable to those of scFv antibodies produced
in E. coli. Recoveries of methamidophos-fortified samples demonstrated that the P. pastoris-derived scFv antibodies can be used to determine the content of methamidophos residue in environmental and agricultural samples.
For our purposes, expression in Pichia proved to be an efficient and economical method for the large-scale production of functional scFv antibodies against methamidophos
for downstream applications. |
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Keywords: | Pichia pastoris ScFv Expression Methamidophos Immunochemical characterization |
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