Production and modification of recombinant L-glutaminase using polyethylene glycol and characterization of physicochemical properties

Abstract

L-glutaminase is an enzyme that hydrolyzes L-glutamine, which can be used as a substitute for asparaginase enzyme in the treatment of ALL cancer. Aim: The purpose of this study is to produce and identify recombinant L-glutaminase from indigenous bacteria and modify it using polyethylene glycol polymer and to investigate its physicochemical properties and then evaluate its anti-leukemia effect. Method: Glutaminase gene (YLM) was cloned in expression vectors. The enzyme was expressed and extracted in the expression system. Air inclusion dissolution was done using urea method and then it was purified using affinity chromatography. Next, in order to optimize the properties of the enzyme, it was attached to polyethylene glycol polymer and its physicochemical properties including kinetic parameters, stability and cytotoxicity were measured and compared with the non-pegylated form. Results: The data obtained from gel electrophoresis show the correctness of PCR, digestion and ligation in all stages of cloning and subcloning, and the analysis of determined sequences also confirmed this issue. Bacterial cultures also confirmed the viability of the transformation. The results of SDS-PAGE confirmed the correctness of induction of expression and extraction of cloned protein in expression vectors. Then, the binding of Peg to the recombinant enzyme was investigated using the results of SDS-PAGE gels and also the results of spectrophotometry, and its binding was confirmed. The results of MTT test showing the sensitivity of Jurkat cells to different doses of pegylated and free enzyme were determined and compared. The data obtained from the examination of apoptosis were also calculated and compared. Conclusion: Currently, PEGylation of recombinant materials with the approach of modifying physicochemical and therapeutic properties such as immunogenicity and short half-life is considered as an effective method in research.