RESUMO
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.
Assuntos
Antineoplásicos , Dickeya chrysanthemi , Leucemia-Linfoma Linfoblástico de Células Precursoras , Animais , Antineoplásicos/metabolismo , Asparaginase/química , Asparagina , Escherichia coli/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , RatosRESUMO
In previous studies Pseudomonas aeruginosal-ASNase complete coding sequence gene, 984 bp (GenBank accession number KU161101.2) was isolated by PCR, cloned into pET28a(+) vector, expressed in E. coli DE3(BL21) pLysS, purified to apparent homogeneity and biochemically characterized. In the present work we highlight large scale production, affinity purification of the recombinant enzyme, effect of osmolytes on the stability of the l-ASNase and cytotoxicity on different cancer cell lines. Successful overexpression was achieved in E. coli as a 6-His-Tag fusion protein after 18 h of induction with lactose at a concentration of 2 g/L in fermentation medium and at 37 °C. The recombinant enzyme was purified to homogeneity using Ni2+ chelated Fast Flow Sepharose resin with 19758.8 specific activity and 10.28 purification fold. With respect to the effect of osmolytes on the stability of the purified enzyme, the majority of the tested osmolytes namely 5% maltose, 5% mannitol, 30% glycerol and 5% BSA were found to increase the stability of the recombinant l-ASNase as compared to the free enzyme. Triple negative breast cancer cell line, MDA-MB-231 treated with recombinant l-ASNase showed significant morphological changes and the IC50 of the purified enzyme was found to be 3.1 IU. Human leukemia cell line, THP-1 treated with l-ASNase showed apoptotic bodies and morphological changes with IC50 of the purified enzyme 1.75 IU. Moreover, the purified recombinant l-ASNase was found to induced cytotoxic effects on colorectal adenocarcinoma cell line, Caco-2 with IC50 of 68.28 IU. Results of apoptosis assay on THP-1 cells revealed that the purified l-ASNase induced early and late apoptosis at 14.16% and 7.56 respectively as compared to the control untreated cells.
Assuntos
Antineoplásicos , Asparaginase , Proteínas de Bactérias , Pseudomonas aeruginosa/genética , Células A549 , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Asparaginase/química , Asparaginase/genética , Asparaginase/isolamento & purificação , Asparaginase/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Células CACO-2 , Escherichia coli/genética , Escherichia coli/metabolismo , Células HCT116 , Humanos , Pseudomonas aeruginosa/enzimologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Células THP-1RESUMO
L-Asparaginase (L-ASNase EC 3.5.1.1) is considered as an important biopharmaceutical drug enzyme in the treatment of childhood acute lymphoblastic leukemia (ALL). In the present study, Pyrococcus furiosus L-ASNase gene was cloned into pET26b (+), expressed in E. coli BL21(DE3) pLysS, and purified to homogeneity using Ni2+ chelated Fast Flow Sepharose resin with 5.7 purification fold and 23.9% recovery. The purified enzyme exhibited a molecular weight of ~33,660 Da on SDS-PAGE and showed maximal activity at 50 °C and pH 8.0. It retained 98.3% and 60.7% initial activity after 60 min at 37 °C and 50 °C, respectively. The recombinant enzyme showed highest substrate specificity towards L-ASNase substrate, while no detectable specificity was observed for l-glutamine, urea, and acrylamide at 10 mM concentration. The Km and Vmax of the purified recombinant enzyme as calculated using Lineweaver-Burk plot were determined to be 1.623 mM and 105 µmol min-1 mg-1, respectively. Human leukemia cell line THP-1 treated with recombinant L-ASNase showed significant morphological changes, and the IC50 of the purified enzyme was found to be 0.8 IU. Moreover, the purified recombinant L-ASNase induced cytotoxic effects on lung adenocarcinoma A549 and colorectal adenocarcinoma Caco-2 cell lines with IC50 of 1.78 IU and 30 IU, respectively.