Utilizing intein-mediated protein cleaving for purification of uricase, a multimeric enzyme
•It is first report on purifying a multimeric therapeutic enzyme with inteins using a well-established and facile system.•E. coli-codon-optimized gene of A. flavus uricase was synthesized and used as the multimeric enzyme.•The pTXB1 vector, containing Mxe GyaA, and chitin binding domain affinity tag...
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Veröffentlicht in: | Enzyme and microbial technology 2016-11, Vol.93-94, p.92-98 |
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Format: | Artikel |
Sprache: | eng |
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Zusammenfassung: | •It is first report on purifying a multimeric therapeutic enzyme with inteins using a well-established and facile system.•E. coli-codon-optimized gene of A. flavus uricase was synthesized and used as the multimeric enzyme.•The pTXB1 vector, containing Mxe GyaA, and chitin binding domain affinity tag was used for cloning the gene.•The purified uricase structural and biochemical characteristics were determined and compared with the uricase purified without using intein.
Uric acid, a side product of nucleotide metabolism, should be cleared from blood stream since its accumulation can cause cardiovascular diseases and gout. Uricase (urate oxidase) converts uric acid to 5-hydroxyisourate, but it is absent in human and other higher apes. Yet, the recombinant form of uricase, Rasburicase, is now commercially available to cure tumor lysis syndrome by lowering serum uric acid level. Developing new methods to efficiently purify pharmaceutical proteins like uricase has attracted researchers’ attention. Self-cleaving intein mediated single column purification is one of these novel approaches. Self-cleaving inteins are modified forms of natural inteins that can excise and join only at one junction site. In this study, the synthetic gene of Aspergillus flavus uricase, a homotetrameric protein, was cloned into pTXB1 vector as a fusion with the N-terminal of Mxe GyrA intein and chitin-binding domain (CBD) for simple purification. Expression was confirmed by western blot analysis. The fusion protein containing uricase-intein-CBD was purified on a chitin column. The cleavage was induced by adding DTT,11Dithiotritol as a reducing agent to release uricase. The purity of uricase and complete excision of the intein and CBD were confirmed by SDS-PAGE22Sodium dodecyl sulfate polyacrylamide gel electrophoresis while its proper folding was proved by circular dichroism and fluorescent emission studies. Isoelectric focusing further confirmed its homogeneity when a single protein band was observed at the predicted pI value. This is the first report of successful purification of a multimeric therapeutic enzyme by intein-mediated protein cleaving using a well-established and facile system. |
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ISSN: | 0141-0229 1879-0909 |
DOI: | 10.1016/j.enzmictec.2016.08.001 |