Expression and Purification of Human Paraoxonase 1 in Pichia pastoris

YU Zi-tong; LIAO Yi-bo; YE Yan-rui

doi:10.13982/j.mfst.1673-9078.2017.7.013

Abstract：Human paraoxonase 1 (hPON1) is a non-specific esterase in human plasma that can hydrolyze various organophosphates,and is considered as an organophosphate pesticide scavenger.In order to obtain high-purity active hPON1,a Pichia pastoris expression system was used to conduct intracellular expression of hPON1.The hPON1 gene was codon-optimized and cloned into the pPICZA expression vector to obtain the recombinant plasmid pPICZA-PON1.The recombinant vector was linearized and transformed into Pichia pastoris X33,and positive transformants were verified by colony polymerase chain reaction (PCR).Batch fermentation of the recombinant strains was performed in shake flasks for 120 h,and the esterase activity ofhPON 1 was 0.15 U/mL.Cells were collected and lysed and the recombinant protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography.Sodium dodecyl sulfate polyacrylamnide gel electrophoresis (SDS-PAGE) results showed that the molecular mass of the recombinant hPON1 was approximately 37 ku,consistent with the expected value.The optimal reaction temperature and pH for the expression of recombinant bPON1 were 45 ℃ and 9.0,respectively.The above results demonstrated that active recombinant hPON1 was successfully expressed,and high purity and active hPON1 was obtained in this study.