L-asparaginase from Dickeya chrysanthemi: expression, purification and cytotoxicity assessment
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 °C and 45 °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.
Faculty of Applied Health Sciences Technology
Life Sciences, Biochemistry, Genetics, Molecular Biology
Indexed in Scopus
Indexed in Web Of Science
Acute lymphoblastic leukemia, cloning, expression, hepatotoxicity, L. asparaginase, pancreatitis
Saeed, Hesham; Elsawy, Eman; Shalaby, Manal; Abdel-Fattah, Manal; Hemida, Asmaa; Eldoksh, Ahmad; Ataya, Farid Shokry; Nematalla, Hesham; Elkewedi, Mohamed; Labrou, Nikolaos N.; and El-Nikhely, Nefertiti, "L-asparaginase from Dickeya chrysanthemi: expression, purification and cytotoxicity assessment" (2021). Faculty of Applied Health Sciences Technology. 151.