Purification, refolding, and characterization of recombinant human paraoxonase-1

Authors : Yeliz DEMİR, Şükrü BEYDEMİR

Pages : 764-776

Doi:10.3906/kim-1501-51

View : 19 | Download : 12

Publication Date : 0000-00-00

Article Type : Research Paper

Abstract :A high density lipoprotein insert ignore into journalissuearticles values(HDL);-linked enzyme with antioxidant and antiatherogenic properties, paraoxonase-1insert ignore into journalissuearticles values(PON1);, prevents the formation of atherosclerotic lesions in humans. In the present study, a recombinant hPON1 gene was produced using a small ubiquitin-related modifier insert ignore into journalissuearticles values(SUMO); fusion protein expression system. To that end, the hPON1 gene was amplified from human liver-ready cDNA, cloned into the expression vector pET SUMO, and expressed in Escherichia coli BL21 insert ignore into journalissuearticles values(DE3);. The predominance of the expressed fusion SUMO-hPON1 protein was inclusion bodies and purified using 6xHis affinity chromatography under natural and denaturing conditions. Subsequently, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein in a single step. The inclusion bodies were solubilized with urea, guanidine hydrochloride, and Triton X-100 and refolded in vitro. After purification, 0.045 mg/mL protein in soluble fraction and 0.108 mg/mL protein from inclusion bodies were obtained. Optimum temperature, pH, and ionic strength for rhPON1 activity were determined as 40 $^{\circ}$C, 10.0, and 100 mM, respectively. The kinetic parameters K$_{m}$ and V$_{\max}$ for rhPON1 were determined as 0.94 mM and 110.01 EU/mL, respectively, by using Lineweaver-Burk plots.
Keywords : Recombinant DNA, cloning, HDL, SUMO expression system, 6xHis affinity chromatography