期刊
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
卷 52, 期 6, 页码 668-680出版社
TAYLOR & FRANCIS INC
DOI: 10.1080/10826068.2021.1983831
关键词
Acute lymphoblastic leukemia; cloning; hepatotoxicity; expression; L; asparaginase; pancreatitis
类别
资金
- Academy of Scientific Research and Technology (ASRT), Egypt [35d/2018]
In this study, the recombinant L-ASNase from Dickeya chrysanthemi (DcL-ASNase) was successfully cloned, expressed, and purified, allowing for analysis of its properties and applications through techniques such as kinetics analysis and immunogenicity evaluation.
Microbial L-asparaginases are aminohydrolases that hydrolyze L-asparagine to L-aspartate. They are used to treat acute lymphoblastic leukemia and Hodgkin's lymphomas and in food industries. Increasing demand for L-ASNases is therefore needed. In the current study, the recombinant L-ASNase from Dickeya chrysanthemi (DcL-ASNase) was cloned into pET28a (+) expression vector and expressed in Escherichia coli as a 6His-tagged fusion protein and purified using Ni2+ chelated Sepharose chromatography resin, yielding a highly purified enzyme. Kinetics analysis allowed the determination of its substrate specificity and the physicochemical parameters that affect enzyme activity. The enzyme showed operational stability at 37 degrees C and 45 degrees C. The immunogenicity of the purified DcL-ASNase was evaluated by measuring the IgG and IgM levels in rats after injection. The cytotoxicity DcL-ASNase in selected cancer cell lines and peripheral blood monocytes was determined. The results showed that the enzyme induces pleiotropic effects, including significant morphological changes and the formation of apoptotic bodies. No cytotoxic effects were observed in peripheral blood monocytes at the same concentrations. In addition, gene expression analysis by RT-PCR of apoptotic biomarkers (Bax, survivin, and Ki-67) allowed the study of the apoptotic mechanism induced by DcL-ASNase on THP-1 cells.
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