Expression And Characterization Of Human Paraoxonase 1 In Pichia Pastoris

Human Paraoxonase 1(hPON1)is a non-specific esterase in human plasma which can hydrolyze various organophosphates.It has been shown that PON1 has the effect of anti-inflammatory,anti-oxidative,anti-diabetic,anti-atherosclerosis and the rescue of organophosphate poisoning.Compared with the existing organic phosphorus antidote,PON1 has better detoxification effect.However,it is so difficult to purify the PON1 from natural sources.Although it has high level of the expression of heterologous protein by E.coli expression system,it always expresses in the form of inclusion bodies and has certain problems in protein folding.Therefore,Pichia pastoris expression system was used to express active hPON1 in this research.Firstly,the hPON1 gene was codon-optimized and cloned into pPICZA expression vector to obtain the recombinant plasmid pPICZA-PON1.The recombinant vector was linearized and transformed into Pichia pastoris X33.Then the positive transformants were verified by colony PCR.Batch fermentation was performed in shake flasks for 120 h,and the enzyme activity of hPON1 was determined as 0.15 U/mL.The recombinant protein was purified by Ni-NTA affinity chromatography.SDS-PAGE showed that the molecular mass of the recombinant hPON1 was approximately 37 kDa which was as expected.Secondly,One-Factor-at-a-Time experiment and Box-Behnken design method was used to optimize the fermentation conditions to improve the expression of rhPON1.The optimum conditions are as followed: Methanol concentration 1.42%,Initial pH value 5.91,and Culture volume 9.39%.The predicted maximum enzyme activity of rhPON1 was 0.737 U/mL.Under the optimal conditions,the enzyme activity of rhPON1 was reached 0.735 U/mL,which was 1.78 times higher than control.The effect of initial induced cell concentration on the expression of rhPON1 were investigated.Increasing the initial induced cell concentration in a certain range can increase the expression of rhPON1.The results could provide the basis for further research.Finally,biochemical characterization of rhPON1 was detected by two different substrates.The Km values of rhPON1 catalyzed by pNPB and paraoxon were 0.2727 mM and 0.4860 mM,respectively.When pNPB was used as substrate,the optimal reaction temperature and pH were 45°C and 9.0,respectively.The enzyme activity of hPON1 was 0.57 U/m L.When paraoxon was used as substrate,the optimal reaction temperature and pH were 45°C and 8.5,respectively.The enzyme activity of hPON1 was 0.17 U/mL.In conclusion,the results demonstrate that the active recombinant h PON1 was successfully expressed and purified in this study.The enzyme activity was improved by 78% than control through optimize its fermentation conditions.When the substrate was pPNB,the enzyme activity was improved by 280% after the characterization studies.When the substrate was paraoxon,the enzyme activity was improved by 42% after the characterization studies.