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3 Biotech

, 8:400 | Cite as

Efficient purification of a recombinant tag-free thermostable Kluyveromyces marxianus uricase by pH-induced self-cleavage of intein and expression in Escherichia coli

  • Bangchun Wang
  • Laipeng Luo
  • Dongmei Wang
  • Rui Ding
  • Jiong Hong
Original Article
  • 67 Downloads

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.

Keywords

Intein Kluyveromyces marxianus Tag free Thermostable Uricase 

Notes

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).

Compliance with ethical standards

Conflict of interest

The authors are aware of the ethical responsibilities and have no conflicts of interest.

Supplementary material

13205_2018_1422_MOESM1_ESM.docx (268 kb)
Supplementary material 1 (DOCX 268 KB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institutes of Life ScienceAnhui Medical UniversityHefeiChina
  2. 2.School of Life ScienceUniversity of Science and Technology of ChinaHefeiChina

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