Abstract
L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells.
Highlights
One of the main therapeutic enzymes of microbial origin, L-asparaginase (L-asparagine amino hydrolase, E.C. 3.5.1.1) is used as a chemotherapeutic agent to treat a number of lymphoproliferative disorders and lymphomas, especially acute lymphoblastic leukemia (ALL) [1,2]
By ascertaining the presence of possible signal sequences in the enzyme’s amino acid sequence, we were able to infer that the first 29 amino acids are very likely to be part of a signal peptide. This is consistent with preliminary results with the enzyme [15], which demonstrated that L-asparaginase from Z. mobilis is a periplasmic enzyme
For the preliminary study of the possible antineoplastic activity of L-asparaginase from Z. mobilis, its nucleotide sequence was aligned with the nucleotide sequences of the L-asparaginases from E. coli and from E. chrysanthemi, since they both have antineoplastic activity and are used in commercial formulations
Summary
One of the main therapeutic enzymes of microbial origin, L-asparaginase (L-asparagine amino hydrolase, E.C. 3.5.1.1) is used as a chemotherapeutic agent to treat a number of lymphoproliferative disorders and lymphomas, especially acute lymphoblastic leukemia (ALL) [1,2]. Lasparaginase works by depleting the exogenous supply of L-asparagine to cells, since malignant. Recombinant L-Asparaginase from Z. mobilis as Antileukemic Agent cells synthesize L-asparagine more slowly than they need and depend on the exogenous supply of this amino acid. Normal cells are able to synthesize all the amino acid they need and are not harmed by the use of L-asparaginase [3,4,5,6]. Type II L-asparaginases from bacteria are homotetramers composed of subunits with approximately 300 amino acids [7]
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