tailieunhanh - Expression, purification, and characterization of recombinant human paraoxonase 1 (rhPON1) in Pichia pastoris

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 pPICZαA vector using a strong AOX promoter, and enzyme secretion in the fermentation medium was achieved by means of Saccharomyces cerevisiae alpha factor signal sequence. | Turkish Journal of Biology Turk J Biol (2015) 39: 649-655 © TÜBİTAK doi: Research Article Expression, purification, and characterization of recombinant human paraoxonase 1 (rhPON1) in Pichia pastoris 1, 1 2 1 Yağmur ÜNVER *, Esabi Başaran KURBANOĞLU , Orhan ERDOĞAN Department of Molecular Biology and Genetics, Faculty of Science, Atatürk University, Erzurum, Turkey 2 Department of Biology, Faculty of Science, Atatürk University, Erzurum, Turkey Received: Accepted/Published Online: Printed: 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 pPICZαA 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 kDa. Biochemical characterization of the enzyme was carried out after purification with a Probond affinity column. The purified paraoxonase 1 activity was determined as U/mL; however, enzyme activity reached U/mL at the end of the characterization studies. According to the results, KM and Vmax values were mM and 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. Key words: Pichia pastoris X-33, hPON1, recombinant protein, purification, characterization 1. Introduction Paraoxonase 1 (PON1), whose activity is related to the toxicology of organophosphorus compounds and cardiovascular health, is a mammalian .

• Sinh học
• Pichia pastoris X 33
• Recombinant protein
• Recombinant human paraoxonase
• Saccharomyces cerevisiae alpha factor signal sequence
• High density lipoprotein