Heterologous expression of recombinant urate oxidase using the intein-mediated protein purification in Pichia pastoris

Abstract

The potential of urate oxidase (uricase) for clinical use has been highlighted because of its role in lowering the blood uric acid levels for the treatment of tumor lysis syndrome. In the present study, the codon-optimized synthetic gene of Aspergillus flavus uricase was fused to the Mxe GyrA intein and chitin-binding domain. The construct was inserted into pPICZA and pPICZαA vectors and electroporated into Pichia pastoris GS115 for the cytosolic and secretory expression. Transformants were screened on gradients of Zeocin up to 2000 μg/ml to find multi-copy integrants. For both constructs, colonies with more resistance were screened for the highest uricase producers by enzyme assay. PCR analysis confirmed successful cassettes insertion into the genome and Mut + phenotype. The gene copy index was determined to be two and five for cytosolic and secretory strains, respectively. Productivity of the cytosolic and secretory strains was found to be 0.74 and 0.001 U/ml culture media in order while the cytosolic recombinant enzyme accounted for about 6% of total proteins. One-step purification of the expressed uricase was done with the aid of the chitin affinity column, followed by DTT induction for intein on-column cleavage. The yield of 40.8 mg/L and K m of 0.22 mM was obtained for intracellular expression. It seems that the intracellular production of uricase can indeed serve as an effective alternative to secretory expression. Moreover, this is the first report considering cytosolic production of uricase using the intein-mediated protein purification in the methylotrophic yeast, P. pastoris.

Economic purification of recombinant uricase by artificial oil bodies

“…The specific activity of the enzyme was increased from 0.53 U/mg in the crude extract to 15.2 U/mg in the purified enzyme, which is comparable with the purified uricases by chromatography columns (Fazel et al 2014 ; Khaleghi and Asad 2021 ).…”

Cloning, Heterologous Expression, and Characterization of a Neutral Uricase from Arthrobacter sp. CSAJ-16 in Cangshan Mountain

“…( Bongaerts et al 1978 ), Arthrobacter sp. ( Suzuki et al 2004 ), Bacillus subtilis ( Khaleghi and Asad 2021 ), Aspergillus flavus ( Li et al 2006 ; Khaleghi and Asad 2021 ), Pseudomonas sp. ( Abdel-Fattah et al 2005 ), Amycolatopsis sp.…”