Production, characterization, and biological impacts of recombinant L- glutaminase on leukemia

Abstract

Introduction: Acute lymphoblastic leukemia (ALL) is the most common type of blood malignancies among children. An essential element of ALL treatment is L-asparaginase. Considering its value, there is an urgent need for an alternative whenever the further use of L-asparaginase is limited. L-glutaminase is a hydrolytic enzyme that can starve tumor cells. Aims of Study: The main aim of this thesis was to produce and characterize the recombinant L-glutaminase from native bacteria and assess its anti-leukemic effect. Method: In the current thesis, Bacillus licheniformis-SL-1 L-glutaminase gene (glsA) was cloned in the pET22b+ and recombinant pET28a+ expression vectors, which was confirmed by PCR experiments using specific primers and sequencing. L-glutaminase was overexpressed in Escherichia coli (BL21 and Origami), extracted by freeze-thawing and sonication techniques, then purified by affinity chromatography. To perform in silico characterization, 3D-modeling and molecular docking methods were implemented. L-glutaminase was characterized via SDS-PAGE, western-blotting, Bradford-assay, and Nessler’s hydrolysis-based activity assay. Finally, the purified enzyme was examined for anti-leukemic activity employing MTT and FITC-labelled-annexin-V assays. Results and Discussion: Gel-electrophoresis data confirmed the complete cloning of a ~1000 bp gene into the host microorganism. There was a 99% similarity between the glsA gene of the native and standard strains. The 3D-modeling predicted that only N-terminal polyhistidine tags are exposed superficially. The SDS-PAGE analysis showed successful soluble overexpression, extraction, and purification of a 36 kDa protein in the Origami expression system. Molecular docking analysis suggested that the 1 glutaminase has a higher affinity for L-asparagine than L-glutamine, which was confirmed by the activity assay. The Km and kcat for L-glutaminase were 39.83 μM and 19.85 s-1. The determined IC50 was ~30μg/mL and apoptosis induction of over 45%. Conclusion: Collectively, the recombinant/active L-glutaminase was successfully produced. The acquired enzyme provided a high anti-leukemic function. If it passes the preclinical evaluations, the engineered enzyme can be considered as an alternative for L-asparaginase.