Volume 21, Issue 1 (2018)
mjms 2018, 21(1): 7-13 |
Back to browse issues page
Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
Azizi M, Enayati S, Salmasizadeh M, Khalaj V. Cloning, Expression, and Purification of Recombinant Urate Oxidase Using Intein Sequence and Elastin-like Protein. mjms 2018; 21 (1) :7-13
URL: http://mjms.modares.ac.ir/article-30-22360-en.html
URL: http://mjms.modares.ac.ir/article-30-22360-en.html
1- Medical Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran
Abstract: (9530 Views)
Aims: Expression and purification of recombinant Urate oxidase as well as other pharmaceutical proteins are a costly process. The use of non-chromatographic methods in the purification process can reduce production costs. So, the aim of this study was cloning, expression, and purification of recombinant urate oxidase, using intein sequence and elastin-like protein (ELP).
Materials and Methods: In the present experimental research, the synthetic urate oxidase gene was cloned in downstream of ELP–Intein in an expression plasmid. After cultivating bacteria containing urate oxidase expression plasmid, the soluble fraction of cell lysate was separated from insoluble portion, using centrifugation. The recombinant urate oxidase was precipitated by adding salt and incubation at 37°C. The resulting precipitant was resuspended in cleavage buffer and the urate oxidase was separated from ELP through Intein moiety. Adding salt for the second time and incubation at 37°C resulted in separation of urate oxidase from fusion partner. After expressing and purifying the urate oxidase, purity and biological activity of recombinant urate oxidase were compared to standard drug.
Findings: The recombinant urate oxidase was expressed and purified using ELP and Intein tags as fusions partners. The rescombinant enzyme showed a purity of %90 equal to 1.89 mg.ml-1 based on protein concentration and 1.64 mg.ml-1 based on activity.
Conclusion: Using Intein sequence and Elastin-like protein, together with the Urate Oxidase enzyme helps to express and purify the enzyme without need for a chromatographic method.
Materials and Methods: In the present experimental research, the synthetic urate oxidase gene was cloned in downstream of ELP–Intein in an expression plasmid. After cultivating bacteria containing urate oxidase expression plasmid, the soluble fraction of cell lysate was separated from insoluble portion, using centrifugation. The recombinant urate oxidase was precipitated by adding salt and incubation at 37°C. The resulting precipitant was resuspended in cleavage buffer and the urate oxidase was separated from ELP through Intein moiety. Adding salt for the second time and incubation at 37°C resulted in separation of urate oxidase from fusion partner. After expressing and purifying the urate oxidase, purity and biological activity of recombinant urate oxidase were compared to standard drug.
Findings: The recombinant urate oxidase was expressed and purified using ELP and Intein tags as fusions partners. The rescombinant enzyme showed a purity of %90 equal to 1.89 mg.ml-1 based on protein concentration and 1.64 mg.ml-1 based on activity.
Conclusion: Using Intein sequence and Elastin-like protein, together with the Urate Oxidase enzyme helps to express and purify the enzyme without need for a chromatographic method.
References
1. Goldman SC, Holcenberg JS, Finklestein JZ, Hutchinson R, Kreissman S, Johnson FL, et al. A randomized comparison between rasburicase and allopurinol in children with lymphoma or leukemia at high risk for tumor lysis. Blood. 2001;97(10):2998-3003. [Link]
2. Pui CH, Jeha S, Irwin D, Camitta B. Recombinant urate oxidase (rasburicase) in the prevention and treatment of malignancy-associated hyperuricemia in pediatric and adult patients: Results of a compassionate-use trial. Leukemia. 2001;15(10):1505-9. [Link] [DOI:10.1038/sj.leu.2402235]
3. FitzGerald O, Fitzpatrick DA, McGeeney KF. Letter: Urate-oxidase treatment for hyperuricaemia. Lancet. 1975;1(7905):525. [Link]
4. Leplatois P, Le Douarin B, Loison G. High-level production of a peroxisomal enzyme: Aspergillus flavus uricase accumulates intracellularly and is active in Saccharomyces cerevisiae. Gene. 1992;122(1):139-45. [Link] [DOI:10.1016/0378-1119(92)90041-M]
5. Giffard M, Ferté N, Ragot F, El Hajji M, Castro B, Bonneté F. Urate oxidase purification by salting-in crystallization: Towards an alternative to chromatography. PLoS One. 2011;6(5):e19013. [Link] [DOI:10.1371/journal.pone.0019013]
6. Chen Z, Wang Z, He X, Guo X, Li W, Zhang B. Uricase production by a recombinant Hansenula polymorpha strain harboring Candida utilis uricase gene. Appl Microbiol Biotechnol. 2008;79(4):545-54. [Link] [DOI:10.1007/s00253-008-1472-8]
7. Li J, Chen Z, Hou L, Fan H, Weng S, Xu C, et al. High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli. Protein Expr Purif. 2006;49(1):55-9. [Link]
8. Pfrimer P, de Moraes LM, Galdino AS, Salles LP, Reis VC, De Marco JL, et al. Cloning, purification, and partial characterization of Bacillus subtilis urate oxidase expressed in Escherichia coli. J Biomed Biotechnol. 2010;2010:674908. [Link]
9. Hershfield MS, Roberts LJ 2nd, Ganson NJ, Kelly SJ, Santisteban I, Scarlett E, et al. Treating gout with pegloticase, a PEGylated urate oxidase, provides insight into the importance of uric acid as an antioxidant in vivo. Proc Natl Acad Sci USA. 2010;107(32):14351-6. [Link]
10. Coiffier B, Mounier N, Bologna S, Fermé C, Tilly H, Sonet A, et al. Efficacy and safety of rasburicase (recombinant urate oxidase) for the prevention and treatment of hyperuricemia during induction chemotherapy of aggressive non-Hodgkin's lymphoma: results of the GRAAL1 (Groupe d'Etude des Lymphomes de l'Adulte Trial on Rasburicase Activity in Adult Lymphoma) study. J Clin Oncol. 2003;21(23):4402-6. [Link]
11. Lan D, Huang G, Shao H, Zhang L, Ma L, Chen Sh, et al. An improved nonchromatographic method for the purification of recombinant proteins using elastin-like polypeptide-tagged proteases. Anal Biochem. 2011;415(2):200-2. [Link]
12. Banki MR, Feng L, Wood DW. Simple bioseparations using self-cleaving elastin-like polypeptide tags. Nat Methods. 2005;2(9):659-61. [Link]
13. Fazel R, Zarei N, Ghaemi N, Namvaran MM, Enayati S, Mirabzadeh Ardakani E, et al. Cloning and expression of Aspergillus flavus urate oxidase in Pichia pastoris. Springerplus. 2014;3:395. [Link]
14. Sambrook J, Russell D. Molecular cloning: A laboratory manual. 1st Volume. 3rd Edition. New York: Cold Spring Harbor Laboratory Press; 2001. [Link]
15. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72(1-2):248-54. [Link] [DOI:10.1016/0003-2697(76)90527-3]
16. Banki MR, Gerngross TU, Wood DW. Novel and economical purification of recombinant proteins: Intein-mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association. Protein Sci. 2005;14(6):1387-95. [Link]
17. Fong BA, Wu WY, Wood DW. Optimization of ELP-intein mediated protein purification by salt substitution. Protein Expr Purif. 2009; 66(2):198-202. [Link] [DOI:10.1016/j.pep.2009.03.009]
18. Fong BA, Wood DW. Expression and purification of ELP-intein-tagged target proteins in high cell density E. coli fermentation. Microb Cell Fact. 2010;19:77. [Link]
19. Shi C, Han TC, Wood DW. Purification of microbially expressed recombinant proteins via a dual ELP split intein system. Methods Mol Biol. 2017;1495:13-25. [Link] [DOI:10.1007/978-1-4939-6451-2_2]
20. Guan D, Chen Z. Affinity purification of proteins in tag-free form: Split intein-mediated ultrarapid purification (SIRP). Methods Mol Biol. 2017;1495:1-12. [Link] [DOI:10.1007/978-1-4939-6451-2_1]
21. Alishah K, Asad S, Khajeh K, Akbari N. Utilizing intein-mediated protein cleaving for purification of uricase, a multimeric enzyme. Enzyme Microb Technol. 2016;93-94:92-8. [Link] [DOI:10.1016/j.enzmictec.2016.08.001]
| Rights and permissions | |
 |
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |