title: Utilizing intein-mediated protein cleaving for purification of uricase, a multimeric enzyme.
abstract: 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,1as a reducing agent to release uricase. The purity of uricase and complete excision of the intein and CBD were confirmed by SDS-PAGE2while 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.
authors.affiliation: Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran.
authors.forename: Khadijeh
authors.initials: K
authors.lastname: Alishah
authors.affiliation: Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran. Electronic address: asad@ut.ac.ir.
authors.forename: Sedigheh
authors.initials: S
authors.lastname: Asad
authors.affiliation: Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran.
authors.forename: Khosro
authors.initials: K
authors.lastname: Khajeh
authors.affiliation: Department of Microbiology, Faculty of Science, Islamic Azad University, Arak, Iran.
authors.forename: Neda
authors.initials: N
authors.lastname: Akbari
mesh_terms: D001231:Aspergillus flavus / Q000201:enzymology / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005800:Genes, Fungal; D005813:Genes, Synthetic; D006801:Humans; D047668:Inteins; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014503:Urate Oxidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D014503:Urate Oxidase
keywords: Aspergillus flavus; Intein; Purification; Uricase; pTXB