Yeliz DEMİR, Şükrü BEYDEMİR

8098

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

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 $^{\circ}$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.
Anahtar Kelimeler:

Recombinant DNA, cloning, HDL, SUMO expression system, 6xHis affinity chromatography

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

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 $^{\circ}$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,

PDF

___

  • Primo-Parmo, S. L.; Sorenson, R. C.; Teiber, J.; La Du, B. N. Genomics 1996, 33, 498–507.
  • Draganov, D. I.; Teiber, J. F.; Speelman, A.; Osawa, Y.; Sunahara, R.; La Du, B. N. J Lipid Res. 2005, 46, 1239–1247.
  • La Du, B. N.; Adkins, S.; Chung-Liang, A. K.; Lipsig, D. Chem. Biol. Interact. 1993, 87, 25–34.
  • Harel, M.; Aharoni, A.; Gaidukov, L.; Brumshtein, B.; Khersonsky, O.; Meged, R.; Dvir, H.; Ravelli, R. B. G.; McCarthy, A.; Toker, L.; et al. Nat. Struct. Mol. Biol. 2004, 11, 412–419.
  • Sorenson, R. C.; Aviram, M.; Bisgaier, C. L.; Billecke, S.; Hsu, C.; La Du, B. N. Chem. Biol. Interact. 1999, 119–120, 243–249.
  • Sorenson, R. C.; Bisgaier, C. L.; Aviram, M.; Hsu, C.; Billecke, S.; La Du, B. N. Arterioscler. Thromb. Vasc. Biol. 1999, 19, 2214–2225.
  • I¸sg¨or, M. M.; Beydemir, S. Eur. J. Pharmacol. 2010, 645, 135–142.
  • Oganesyan, N.; Kim, S. H.; Kim, R. J. Struct. Funct. Genomics 2005, 6, 177–182.
  • Baneyx, F.; Mujacic, M. Nat. Biotechnol. 2004, 22, 1399–1408.
  • Kane, J. F.; Hartley, D. L. Trends Biotechnol. 1988, 6, 95–101.
  • Patra, A. K.; Mukhopadhyay, R.; Mukhija, R.; Krishnan, A.; Garg, L. C.; Panda, A.K. Protein Expr. Purif. 2000, 18, 182–192.
  • Fahnert, B.; Lilie, H.; Neubauer, P. Adv. Biochem. Eng. Biotechnol. 2004, 89, 93–142.
  • Rudolph, R.; Lilie, H. FASEB J. 1996, 10, 49–56.
  • Vallejo, L. F.; Rinas, U. Microb. Cell Fact. 2004, 3, 2–12.
  • Clark, E. D. Curr. Opin. Biotechnol. 1998, 9, 157–163.
  • Lilie, H.; Schwarz, E.; Rudolph, R. Curr. Opin. Biotechnol. 1998, 9, 497–501.
  • Khan, R. H.; Rao, K. B.; Eshwari, A. N.; Totey, S. M.; Panda, A. K. Biotechnol. Prog. 1998, 14, 722–728.
  • Singh, S. M.; Panda, A. K. J. Biosci. Bioeng. 2005, 99, 303–310.
  • Heiker, J. T.; Kl¨oting, N.; Bl¨uher, M.; Beck-Sickinger, A. G. Biochem. Biophys. Res. Commun. 2010, 398, 32–37.
  • Tsumoto, K.; Umetsu, M.; Kumagai, I.; Ejima, D.; Arakawa, T. Biochem. Biophys. Res. Commun. 2003, 312, 1383–1386.
  • Kudou, M.; Yumioka, R.; Ejima, D.; Arakawa, T.; Tsumoto, K. Protein Expr. Purif. 2011, 75, 46–54.
  • Datar, R. V.; Cartwright, T.; Rosen, C. G. Biotechnology (NY) 1993, 11, 349–357.
  • Ekinci, D.; Beydemir, S. Biol. Trace Elem. Res. 2010, 135, 112–120.
  • Ekinci, D.; Sent¨urk, M.; Beydemir, S.; K¨ufrevio˘glu, O. I.; Supuran, C. T. Chem. Biol. Drug Des. 2010, 76, 552–558.
  • T¨urke¸s, C.; S¨oy¨ut, H.; Beydemir, S. Pharmacol. Rep. 2014, 66, 74–80.
  • Bradford, M. M. Anal. Biochem. 1976, 72, 248–254.
  • Gen¸cer, N.; Arslan, O. J Chromatogr. B Analyt. Technol Biomed. Life Sci. 2009, 877, 134–140.
  • Ates, O.; Azizi, S.; Alp, H. H.; Kiziltunc, A.; Beydemir, S.; Cinici, E.; Kocer, I.; Baykal, O. Tohoku J. Exp. Med. 2009, 217, 17–22.
  • Aviram, M.; Rosenblat, M.; Bisgaier, C. L.; Newton, R.S.; Primo-Parmo, S. L.; La Du, B. N. J. Clin. Invest. 1998, 1, 1581–1590.
  • Aviram, M.; Rosenblat, M.; Bisgaier, C. L. J. Clin. Invest. 1998, 101, 2215–2257.
  • La Du, B. N. In Pharmacogenetics of Drug Metabolism; Kalow, W., Ed. Pergamon Press: New York, NY, USA, 1992, pp 51–91.
  • Alici, H. A.; Ekinci, D.; Beydemir, S. Clin. Biochem. 2008, 41, 1384–390.
  • Mackness, M. I.; Durrington, P. N. Atherosclerosis 1995, 115, 243–253.
  • Furlong, C. E.; Costa, L. G.; Hassett, C.; Richter, R. J.; Sundstrom, J. A.; Adler, D. A.; Disteche, C. M.; Omiecinski, C. J.; Chapline, C.; Crabb, J. W.; et al. Chem. Biol. Interact. 1993, 87, 35–48.
  • Avcikurt, A. S.; Sinan, S.; Kockar, F. J. Enzyme Inhib. Med. Chem. 2014, 17, 1–5.
  • Lu, H.; Zhu, J.; Zang, Y.; Ze, Y.; Qin, J. Protein Expr. Purif. 2006, 46, 92–99.
  • Holler, C.; Vaughan, D.; Zhang, C. J. Chromatogr. A 2007, 142, 98–105.
  • Malakhov, M. P.; Mattern, M. R.; Malakhova, O. A.; Drinker, M.; Weeks, S. D.; Butt, T. R. J. Struct. Funct. Genomics 2004, 5, 75–86.
  • Stevens, R. C.; Suzuki, S. M.; Cole, T. B.; Park, S. S.; Richter, R. J.; Furlong, C. E. Proc. Natl. Acad. Sci. USA 2008, 105, 12780–12784.
  • Gopal, G. J.; Kumar, A. Protein J. 2013, 32, 419–425.
  • Josse, D.; Xie, W.; Renault, F.; Rochu, D.; Schopfer, L. M.; Masson, P.; Lockridge, O. Biochemistry 1999, 38, 2816–2825.
  • Aharoni, A.; Gaidukov, L.; Yagur, S.; Toker, L.; Silman, I.; Tawfik, D. S. Proc. Natl. Acad. Sci. USA 2004, 101, 482–487.
  • Sarkar, M.; Harsch, C. K.; Matic, G. T.; Hoffman, K.; Norris, J. R.; Otto, T. C.; Lenz, D. E.; Cerasoli, D. M.; Magliery, T. J. Journal of Lipids 2012, 2012, 610937.
  • Suzuki, S. M.; Stevens, R. C.; Richter, R. J.; Cole, T. B.; Park, S.; Otto, T. C.; Cerasoli, D. M.; Lenz, D. E.; Furlong, C. E. Adv. Exp. Med. Biol. 2010, 660, 37–45.
  • Bajaj, P.; Tripathy, R. K.; Aggarwal, G.; Pande, A. H. Scientific World Journal. 2014, 2014, 854391.
  • Renault, F.; Chabri`ere, E.; Andrieu, J. P.; Dublet, B.; Masson, P.; Rochu, D. J. Chromatogr. B 2006, 836, 15–21.
  • Laemmli, U. K. Nature 1970, 227, 680–685.
  • Lineweaver, H.; Burk, D. J. Am. Chem. Soc. 1934, 56, 658–661.
Turkish Journal of Chemistry-Cover
  • ISSN: 1300-0527
  • Yayın Aralığı: 6
  • Yayıncı: TÜBİTAK
Arşiv
Sayıdaki Diğer Makaleler

Synthesis and biological evaluation of a new series of 4-alkoxy-2-arylquinoline derivatives as potential antituberculosis agents

Gonca TOSUN, Tayfun ARSLAN, Zeynep İSKEFİYELİ, Murat KÜÇÜK, Şengül Alpay KARAOĞLU, Nurettin YAYLI

Microwave-assisted synthesis of novel 8-chloro-[1,2,4]triazolo[4,3-a]pyridine derivatives

Ming-Yan YANG, Lin-Jiong ZHANG, Zhao-Hui SUN, Xing-Hai LIU, Jian-Quan WENG, Cheng-Xia TAN, Bei-Zhen HU

A water-soluble perylene derivative for live-cell imaging

Yongshan MA, Fengxia ZHANG, Jinfeng ZHANG, Tianyi JIANG

Synthesis and characterization of novel urea and thiourea substitute cyclotriphosphazene compounds as naked-eye sensors for F − and CN− anions

Özgür ÖZAY, Mehmet YILDIRIM, Hava OZAY

Enhanced electrocatalytic efficiency of C/MWNTs for methanol oxidation using Ni deposited on MWNTs

Murat FARSAK, Gülfeza KARDAŞ

Simultaneous analysis of losartan, its active metabolite, and hydrochlorothiazide in human plasma by a UPLC-MS/MS method

Priyanka A. SHAH, Primal SHARMA, Jaivik V. SHAH, Mallika SANYAL, Pranav S. SHRIVASTAV

A comparative study on fabrication of Cu$_{2}$ZnSnS$_{4}$ (CZTS) nanofibers using acetate and chloride metal precursors

BURAK ZAFER BÜYÜKBEKAR, FARUK ÖZEL, HÜSEYİN ŞAKALAK, HALİT ÇAVUŞOĞLU, MUSTAFA ERSÖZ, MAHMUT KUŞ, MUSTAFA SELMAN YAVUZ

Optimization of the adsorption of a textile dye onto nanoclay using a central composite design

Aydin HASSANI, Murat KIRANŞAN, Reza Darvishi Cheshmeh SOLTANI, Alireza KHATAEE, Semra KARACA

Synthesis of metal-free and metallophthalocyanines containing 18- and 21-membered macrocycles with mixed donor atoms and their metal-ion binding properties

Halil Zeki GÖK

Palladium nanoparticles supported on modified polystyrene resin as a polymeric catalyst for Sonogashira Hagihara coupling reactions

Soheila GHASEMI, Fatemeh DODEJI NOWROOZI, Bahman TAMAMI