Abstract
Uricase as an important healthcare-related protein is extensively used in the treatment of tumor lysis syndrome and in the manufacture of serum uric-acid diagnostic kits. In this study, a gene of a new thermostable uricase (KmUOX) was cloned from thermotolerant yeast Kluyveromyces marxianus. The uricase was fused with a self-cleaving intein and cellulose-binding affinity tag and expressed in Escherichia coli BL21 (DE3). Through the binding to inexpensive cellulose and in situ intein cleavage induced by a pH change, tag-free uricase (KmUOX) was efficiently purified with a 77.11% yield via a single-step column purification strategy. This tag-free uricase showed Km, Vmax, and Kcat values of 67.60 µM, 56.35 µM/(min mg), and 32.74 S−1, respectively. Furthermore, this pure uricase was relatively thermostable and retained 79.75% of activity when incubated at 40 °C for 90 h. Thus, this pH-induced self-cleavable intein system combined with a cellulose matrix for affinity chromatography is proven here to be an effective and low-cost method for recombinant-uricase purification. Moreover, the stability of KmUOX makes it useful for clinical applications.
Similar content being viewed by others
References
Alishah K, Asad S, Khajeh K, Akbari N (2016) Utilizing intein-mediated protein cleaving for purification of uricase, a multimeric enzyme. Enzyme Microb Technol 93–94:92–98
Arnau J, Lauritzen C, Petersen GE, Pedersen J (2006) Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Exp Purif 48(1):1–13
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
Cammalleri L, Malaguarnera M (2007) Rasburicase represents a new tool for hyperuricemia in tumor lysis syndrome and in gout. Int J Med Sci 4(2):83–93
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(4):545–554
Chu R, Lin Y, Usuda N, Rao MS, Reddy JK, Yeldandi AV (1996) Mutational analysis of the putative copper-binding site of rat urate oxidase. Ann N Y Acad Sci 804:781–786
Fazel R, Zarei N, Ghaemi N, Namvaran MM, Enayati S, Mirabzadeh Ardakani E, Azizi M, Khalaj V (2014) Cloning and expression of Aspergillus flavus urate oxidase in Pichia pastoris. Spring Plus 3:395
Hong J, Wang Y, Ye X, Zhang YH (2008a) Simple protein purification through affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage. J Chromatogr A 1194(2):150–154
Hong J, Ye XH, Wang YR et al (2008b) Bioseparation of recombinant cellulose-bindning module-proteins by affinity adsorption on an ultra-high-capacity cellulosic adsorbent. Anal Chim Acta 621(2):193–199
Imani M, Shahmohamadnejad S (2017) Recombinant production of Aspergillus flavus uricase and investigation of its thermal stability in the presence of raffinose and lactose. 3 Biotech 7(3):201
Kai L, Ma XH, Zhou XL, Jia XM, Xia L, Guo KP (2008) Purification and characterization of a thermostable uricase from Microbacterium sp. strain ZZJ4-1. World J Microbiol Biotechnol 24(3):401–406
Kotb E (2015) Characterization of a thermostable uricase isolated from Bacillus firmus DWD-33 and its application for uric acid quantification in human serum. Protein Pept Lett 22(5):402–409
Koyama Y, Ichikawa T, Nakano E (1996) Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding the Candida utilis urate oxidase (uricase). J Biochem 120(5):969–973
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685
Legoux R, Delpech B, Dumont X, Guillemot JC, Ramond P, Shire D, Caput D, Ferrara P, Loison G (1992) Cloning and expression in Escherichia coli of the gene encoding Aspergillus flavus urate oxidase. J Biol Chem 267(12):8565–8570
Li J, Chen Z, Hou L, Fan H, Weng S, Xu C, Ren J, Li B, Chen W (2006) High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli. Protein Expr Purif 49(1):55–59
Li W, Xu S, Zhang B, Zhu Y, Hua Y, Kong X, Sun L, Hong J (2017) Directed evolution to improve the catalytic efficiency of urate oxidase from Bacillus subtilis. PLoS One 12(5):e0177877
Liu JG, Li GX, Liu H et al (1994) Purification and properties of uricase from Candida Sp and its application in uric acid analysis in serum. Appl Biochem Biotechnol 47(1):57–63
Liu X, Wen M, Li J, Zhai F, Ruan J, Zhang L, Li S (2011) High-yield expression, purification, characterization, and structure determination of tag-free Candida utilis uricase. Appl Microbiol Biotechnol 92(3):529–537
Lotfy WA (2008) Production of a thermostable uricase by a novel Bacillus thermocatenulatus strain. Biores Technol 99(4):699–702
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(3):426–429
Patel KS, Lau JE, Zembillas AS et al (2017) Single 4.5 mg fixed-dose of rasburicase for hyperuricemia associated with tumor lysis syndrome. J Oncol Pharm Pract 23(5):333–337
Pfrimer P, de Moraes LM, Galdino AS, Salles LP, Reis VC, De Marco JL, Prates MV, Bloch C Jr, Torres FA (2010) Cloning, purification, and partial characterization of Bacillus subtilis urate oxidase expressed in Escherichia coli. J Biomed Biotechnol 2010:674908
Pui CH, Mahmoud HH, Wiley JM, Woods GM, Leverger G, Camitta B, Hastings C, Blaney SM, Relling MV, Reaman GH (2001) Recombinant urate oxidase for the prophylaxis or treatment of hyperuricemia in patients With leukemia or lymphoma. J Clin Oncol Off J Am Soc Clin Oncol 19(3):697–704
Ravichandran R, Hemaasri S, Cameotra SS, Jayaprakash NS (2015) Purification and characterization of an extracellular uricase from a new isolate of Sphingobacterium thalpophilum (VITPCB5). Protein Expr Purif 114:136–142
Shaaban MI, Abdelmegeed E, Ali YM (2015) Cloning, expression, and purification of recombinant uricase enzyme from Pseudomonas aeruginosa Ps43 using Escherichia coli. J Microbiol Biotechnol 25(6):887–892
Suzuki K, Sakasegawa S, Misaki H, Sugiyama M (2004) Molecular cloning and expression of uricase gene from Arthrobacter globiformis in Escherichia coli and characterization of the gene product. J Biosci Bioeng 98(3):153–158
Wan W, Wang D, Gao X, Hong J (2011) Expression of family 3 cellulose-binding module (CBM3) as an affinity tag for recombinant proteins in yeast. Appl Microbiol Biotechnol 91(3):789–798
Wang D, Hong J (2014) Purification of a recombinant protein with cellulose-binding module 3 as the affinity tag. Methods Mol Biol 1177:35–45
Acknowledgements
This work was supported by the National Natural Science Foundation of China (31570082 and 31770085), Anhui Provincial Natural Science Foundation (1608085MC47), Natural Science Research Project of the Education Department of Anhui Province (KJ2015A042), and National Undergraduate Training Programs for Innovation and Entrepreneurship (201510366040).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors are aware of the ethical responsibilities and have no conflicts of interest.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Wang, B., Luo, L., Wang, D. et al. Efficient purification of a recombinant tag-free thermostable Kluyveromyces marxianus uricase by pH-induced self-cleavage of intein and expression in Escherichia coli. 3 Biotech 8, 400 (2018). https://doi.org/10.1007/s13205-018-1422-9
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s13205-018-1422-9