Highly efficient Pyrococcus furiosus recombinant L-asparaginase with no glutaminase activity: Expression, purification, functional characterization, and cytotoxicity on THP-1, A549 and Caco-2 cell lines
| Autor: | Manal Abdel-Fattah, Nikolaos E. Labrou, Hesham Saeed, Farid S. Ataya, Manal Shalaby, Ahmed Hussein, Ahmad Eldoksh, Nefertiti El-Nikhely, Hisham Nematalla, Asmaa Hemida, Nihal Aly |
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| Rok vydání: | 2020 |
| Předmět: |
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Molecular Protein Conformation Gene Expression Antineoplastic Agents Apoptosis 02 engineering and technology Hemolysis Biochemistry Glutaminase activity Substrate Specificity law.invention Sepharose 03 medical and health sciences Structural Biology law Cell Line Tumor Asparaginase Humans THP1 cell line Amino Acid Sequence Molecular Biology 030304 developmental biology chemistry.chemical_classification 0303 health sciences Base Sequence Molecular Structure biology Chemistry Substrate (chemistry) General Medicine Hydrogen-Ion Concentration 021001 nanoscience & nanotechnology biology.organism_classification Molecular biology Recombinant Proteins Enzyme Activation Pyrococcus furiosus Enzyme Cell culture Recombinant DNA Caco-2 Cells 0210 nano-technology |
| Zdroj: | International Journal of Biological Macromolecules. 156:812-828 |
| ISSN: | 0141-8130 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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