Utilizing intein-mediated protein cleaving for purification of uricase, a multimeric enzyme

摘要

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,(1) as a reducing agent to release uricase. The purity of uricase and complete excision of the intein and CBD were confirmed by SDS-PAGE(2) 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. (C) 2016 Elsevier Inc. All rights reserved.