Akademik Veri Yönetim Sistemi
TURKISH JOURNAL OF CHEMISTRY, cilt.39, sa.4, ss.764-776, 2015 (SCI-Expanded)
A high density lipoprotein (HDL)-linked enzyme with antioxidant and antiatherogenic properties, paraoxonase-1(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 (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 (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 degrees 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.