Production of Recombinant E.coli Asparaginase on a Laboratory Scale

Abstract

Background: L-asparaginase is an enzyme derived from E. coli and is one of the drugs used to treat acute lymphoblastic leukemia. Decreased blood asparagine after the use of the asparaginase can lead to death for these cells. The aim of this project is to investigate the possibility of ehtproduction and purification of ehtasparaginase enzyme. Methods: The asparaginase gene of E. coli was amplified using PCR technique. After purification, the gene was cloned into pET28a vector and transformed into a bacterial host. After transformation, different protein expression conditions were investigated for optimization. Recombinant enzyme expression was confirmed by SDS-PAGE and Western blotting. The asparaginase enzyme was then purified by affinity chromatography using a column containing Ni-NTA resin and a hybrid method and finally, its enzymatic activity was investigated. Findings: The high production of asparaginase enzyme in E. coli was at 37 °C, 1 mM concentration of IPTG, and incubation 20 hours after induction. Insoluble asparaginase enzyme was purified using a Ni-NTA column and the refolding process was performed on the column. According to the activity of the refolded enzyme in the enzyme activity test, it can be concluded that the refolding process of the enzyme has been done correctly. Conclusion: Due to the great importance of asparaginase production in Iran and the need of a group of patients to use this enzyme, in this project, by optimizing the expression and purification process, the L-asparaginase enzyme was produced on a laboratory scale. © 2023 Isfahan University of Medical Sciences(IUMS). All rights reserved.