Expression, purification, and characterization of recombinant humanparaoxonase 1 (rhPON1) in Pichia pastoris

Abstract

The main purpose of the present study was to perform the expression in Pichia pastoris X-33 of the human paraoxonase 1 (hPON1) enzyme, which is a mammalian serum protein. Extracellular hPON1 enzyme was expressed with the pPICZ?A vector using a strong AOX promoter, and enzyme secretion in the fermentation medium was achieved by means of Saccharomyces cerevisiae alpha factor signal sequence. The recombinant cells were grown in a shaking flask containing production medium. SDS-PAGE and Western blot analysis illustrated that the molecular mass of extracellular hPON1 enzyme produced by the recombinant P. pastoris strain was 59.1 kDa. Biochemical characterization of the enzyme was carried out after purification with a Probond affinity column. The purified paraoxonase 1 activity was determined as 18.9 U/mL; however, enzyme activity reached 31.27 U/mL at the end of the characterization studies. According to the results, KM and Vmax values were 0.025 mM and 28.4 U/mL, respectively, in 100 mM glycine-NaOH buffer (pH 10) containing 2 mM Ca2+ at 15 °C. This is the first report on the expression and production of hPON1 in P. pastoris.