Isolation Cloning Purification And Properties Of Urate Oxidase From Aspergillus Flavus

Abstract

newline ABSTRACT newlineEvolution of Recombinant DNA technology led to the procession of therapeutics in oncology autoimmune diabetic cardiovascular diagnosis and in many other sections of industrial platforms Demand for these recombinant protein products are increasing day by day and created the need to develop efficient expression techniques to increase the productivity with low production cost The key factors in the development of therapeutic biologics are starting the work with authenticated gene sequence and cost effective process development Urate Oxidase the enzyme from Aspergillus flavus is cloned in Escherichia coli and used in the prevention and treatment of hyperuricemia associated with lymphoid malignancies Here the soil microbe capable of producing urate oxidase was isolated and identified An optimized workflow for the cloning and high volumetric production of recombinant urate oxidase in E coli with systematic study of the expression levels was depicted The Escherichia coli strain Rosetta was used as the workhorse for production of recombinant Urate Oxidase In the current work high expression levels forty percent with the highest wet pellet of sixty gram per liter was attained Parameters including confirmation of the gene orientation and other upstream parameters such as suitable OD to induce the protein optimal IPTG concentration for induction and time to harvest the expressed protein were studied systematically to achieve the high volumetric productivity In the present work an effective and an efficient purification process for improving the recovery and quality of recombinant Urate oxidase expressed in Escherichia coli by designing various types of chromatography techniques like ion exchange chromatography hydrophobic interaction chromatography and Multimodal chromatography in various combinations was successfully developed In the process one point one two gram per liter of pure urate oxidase was obtained The overall chromatogrpahy yield recovery is 70 percent and the purity obtained is 99 percent The yield and purity obtained by the process is the higher than any other processes reported and published literature newlineThe biological market is increasing day by day so the regulatory bodies are becoming more stringent to maintain the quality of the product To meet the highest quality standards it is necessary to assess the quality of the product in each and every step of the biotherapeutic drug manufacturing Various methods for the determination of its molecular weight structural integrity invitro bioassay and other methods were employed to compare Urate Oxidase with the standard Integrity by DSS cross linking is first of its kind for Urate Oxidase expressed in Escherichia coli newline newline