Journal of Microbiology and Biotechnology
The Korean Society for Microbiology and Biotechnology publishes the Journal of Microbiology and Biotechnology.

2015 ; Vol.25-6: 887~892

AuthorMona I. Shaaban, Eman Abdelmegeed, Youssif M. Ali
Place of dutyMicrobiology Department, Faculty of Pharmacy, Mansoura University, Egypt
TitleCloning, Expression, and Purification of Recombinant Uricase Enzyme from Pseudomonas aeruginosa Ps43 Using Escherichia coli
PublicationInfo J. Microbiol. Biotechnol.2015 ; Vol.25-6
AbstractUricase is an important microbial enzyme that can be used in the clinical treatment of gout, hyperuricemia, and tumor lysis syndrome. A total of 127 clinical isolates of Pseudomonas aeruginosa were tested for uricase production. A Pseudomonas strain named Ps43 showed the highest level of native uricase enzyme expression. The open reading frame of the uricase enzyme was amplified from Ps43 and cloned into the expression vector pRSET-B. Uricase was expressed using E. coli BL21 (DE3). The ORF was sequenced and assigned GenBank Accession No. KJ718888. The nucleotide sequence analysis was identical to the coding sequence of uricase gene puuD of P. aeruginosa PAO1. We report the successful expression of P. aeruginosa uricase in Escherichia coli. E. coli showed an induced protein with a molecular mass of about 58 kDa that was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. We also established efficient protein purification using the Ni-Sepharose column with activity of the purified enzyme of 2.16 IU and a 2-fold increase in the specific activity of the pure enzyme compared with the crude enzyme.
Full-Text
Key_wordUricase, P. aeruginosa, expression, Western blot, recombinant
References
  1. Adamek V, Kralova B, Suchova M, Valentova O, Demnerova K. 1989. Purification of mic robial uricase. J. Chromatogr. 497:268-275.
    CrossRef
  2. Ali UF, Ibrahim ZM. 2013. Effect of irradiation on uricase produced by two strains of Asperigillus niger. Life Sci. J. 10:962-967.
  3. Anderson A, Vijayakumar S. 2012. Isolation and optimization of Pseudomonas aeruginosa for uricase production. Int. J. Pharma Biol. Sci. 3: 143-150.
  4. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
    CrossRef
  5. Cammalleri L, Malaguarnera M. 2007. Rasburicase represents a new tool for hyperuricemia in tumor lysis syndrome and in gout. Int. J. Med. Sci. 4: 83-93.
    Pubmed CrossRef Pubmed Central
  6. Chen M, Cai L, Fang Z, Tian H, Gao X, Yao W. 2008. Sitespecific incorporation of unnatural amino acids into urate oxidase in Escherichia coli. Protein Sci. 17: 1827-1833.
    Pubmed CrossRef Pubmed Central
  7. Chen Z, Wang Z, He X, Guo X, Li W, Zhang B. 2008. Uricase production by a recombinant Hansenula polymorpha strain harboring Candida utilis uricase gene. Appl. Microbiol. Biotechnol. 79: 545-554.
    Pubmed CrossRef
  8. Chohan S, Becker MA. 2009. Update on emerging uratelowering therapies. Curr. Opin. Rheumatol. 21: 143-149.
    Pubmed CrossRef
  9. Colloc’h N, el Hajji M, Bachet B, L’Hermite G, Schiltz M, Prange T, et al. 1997. Crystal structure of the protein drug urate oxidase-inhibitor complex at 2.05 A resolution. Nat. Struct. Biol. 4: 947-952.
    Pubmed CrossRef
  10. Elmer K, Washington W, Jr Stephen A, William J, Gary P, Paul S, Gail W. 2006. Koneman's Color Atlas and Textbook of Diagnostic Microbiology, 6th Ed. Lippincott Williams and Wilkins, London.
    Pubmed Central
  11. Essam AA, Ali M, Fareed M. 2005. Studies on uricase induction in certain bacteria. Egyptian J. Biol. 7: 44-54.
  12. Fazel R, Zarei N, Ghaemi N, Namvaran MM, Enayati S, Mirabzadeh Ardakani E, et al. 2014. Cloning and expression of Aspergillus flavus urate oxidase in Pichia pastoris. Springerplus 3: 395.
    Pubmed CrossRef Pubmed Central
  13. Feig DI, Kang D H, J ohnson R J. 2 008. U ric acid a nd cardiovascular risk. N. Engl. J. Med. 359: 1811-1821.
    Pubmed CrossRef Pubmed Central
  14. George RL, Jr Sundy JS. 2012. Pegloticase for treating refractory chronic gout. Drugs Today (Barc). 48: 441-449.
  15. Hogardt M, Heesemann J. 2010. Adaptation of Pseudomonas aeruginosa during persistence in the cystic fibrosis lung. Int. J. Med. Microbiol. 300: 557-562.
    Pubmed CrossRef
  16. Hummel M, Buchheidt D, Reiter S, Bergmann J, Adam K, Hehlmann R. 2005. Recurrent chemotherapy-induced tumor lysis syndrome (TLS) with renal failure in a patient with chronic lymphocytic leukemia - successful treatment and prevention of TLS with low-dose rasburicase. Eur. J. Haematol. 75: 518-521.
    Pubmed CrossRef
  17. Ishikawa J, Yamashita A, Mikami Y, Hoshino Y, Kurita H, Hotta K, et al. 2004. The complete genomic sequence of Nocardia farcinica IFM 10152. Proc. Natl. Acad. Sci. USA 101:14925-14930.
    Pubmed CrossRef Pubmed Central
  18. Jagger KS, Bahner DR, Warren RL. 1983. Protease phenotypes of Pseudomonas aeruginosa isolated from patients with cystic fibrosis. J. Clin. Microbiol. 17: 55-59.
    Pubmed Pubmed Central
  19. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
    Pubmed CrossRef
  20. Lehejckova R, Demnerova K, Kralova B. 1986. Screening of microorganisms with uricase activity. Biotechnol. Lett. 8: 341-342.
    CrossRef
  21. Li J, Chen Z, Hou L, Fan H, Weng S, Xu C, et al. 2006. High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli. Protein Expr. Purif. 49: 55-59.
    Pubmed CrossRef
  22. Lyczak JB, Cannon CL, Pier GB. 2000. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect. 2: 1051-1060.
    CrossRef
  23. Nishiya Y, Hibi T, Oda J. 2002. A purification method of the diagnostic enzyme Bacillus uricase using magnetic beads and non-specific protease. Protein Expr. Purif. 25: 426-429.
    CrossRef
  24. Oda M, Satta Y, Takenaka O, Takahata N. 2002. Loss of urate oxidase activity in hominoids and its evolutionary implications. Mol. Biol. Evol. 19: 640-653.
    Pubmed CrossRef
  25. Pfrimer P, de Moraes LM, Galdino AS, Salles LP, Reis VC, De Marco JL, et al. 2010. Cloning, purification, and partial characterization of Bacillus subtilis urate oxidase expressed in Escherichia coli. J. Biomed. Biotechnol. 2010: 674908.
    Pubmed CrossRef Pubmed Central
  26. Pui CH, Mahmoud HH, Wiley JM, Woods GM, Leverger G, Camitta B, et al. 2001. Recombinant urate oxidase for the prophylaxis or treatment of hyperuricemia in patients with leukemia or lymphoma. J. Clin. Oncol. 19: 697-704.
    Pubmed
  27. Richette P, Bardin T. 2010. Gout. Lancet 375: 318-328.
    CrossRef
  28. Saeed HM, Abdel-Fattah YR, Gohar YM, Elbaz MA. 2004. Purification and characterization of extracellular Pseudomonas aeruginosa urate oxidase enzyme. Pol. J. Microbiol. 53: 45-52.
    Pubmed
  29. Schiavon O, Caliceti P, Ferruti P, Veronese FM. 2000. Therapeutic proteins: a comparison of chemical and biological properties of uricase conjugated to linear or branched poly (ethylene glycol) and poly (N-acryloyl morpholine). Farmaco 55: 264-269.
    CrossRef
  30. Shin HY, Kang HJ, Park ES, Choi HS, Ahn HS, Kim SY, et al. 2006. Recombinant urate oxidase (rasburicase) for the treatment of hyperuricemia in pediatric patients with hematologic malignancies: results of a compassionate prospective multicenter study in Korea. Pediatr. Blood Cancer 46: 439-445.
    Pubmed CrossRef
  31. SitiHatijah PM, Wan Ruhayu WR. 2013. Preliminary study on factors that enhanced the production of uricase by Aspergillus flavus. Int. J. Biosci. Biochem. Bioinform. 3: 513-515.
  32. Vogt B. 2005. Urate oxidase (rasburicase) for treatment of severe tophaceous gout. Nephrol. Dial. Transplant. 20: 431-433.
    Pubmed CrossRef
  33. Woods DE, Schaffer MS, Rabin HR, Campbell GD, Sokol PA. 1986. Phenotypic comparison of Pseudomonas aeruginosa strains isolated from a variety of clinical sites. J. Clin. Microbiol. 24: 260-264.
    Pubmed Pubmed Central
  34. Wretlind B, Pavlovskis OR. 1983. Pseudomonas aeruginosa elastase and its role in Pseudomonas infections. Rev. Infect. Dis. 5: S998-S1004.
    Pubmed CrossRef
  35. Zhang J, Ren J, Li B, Liu S, Hou L, Fu L, et al. 2010. Construction, expression, purification and characterization of mutant of Aspergillus flavus urate oxidase. Sheng Wu Gong Cheng Xue Bao 26: 1102-1107.
    Pubmed
  36. Zhang P, Lu X, Li Q, Lin X, Liu H, Ma X. 2012. Cloning and characterization of a thermostable urate oxidase from Microbacterium sp. strain ZZJ4-1. Sheng Wu Gong Cheng Xue Bao 28: 813-822.
    Pubmed



Copyright © 2009 by the Korean Society for Microbiology and Biotechnology.
All right reserved. Mail to jmb@jmb.or.kr
Online ISSN: 1738-8872    Print ISSN: 1017-7825    Powered by INFOrang.co., Ltd