Expression, characterization and cytotoxicity of recombinant l-asparaginase II from Salmonella paratyphi cloned in Escherichia coli.

Autor: Abdullah EM; Department of Biochemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Riyadh, Saudi Arabia., Khan MS; Department of Biochemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Riyadh, Saudi Arabia., Aziz IM; Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia., Alokail MS; Department of Biochemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Riyadh, Saudi Arabia., Karthikeyan S; Centre for Healthcare Advancement, Innovation and Research, Vellore Institute of Technology University, Chennai Campus, Chennai 600127, India., Rupavarshini M; Division of Physics, School of Advanced Sciences, Vellore Institute of Technology, Chennai Campus, Vandalur - Kelambakkam Road, Chennai, Tamil Nadu 600127, India., Bhat SA; College of Science, Cluster University Srinagar, India., Ataya FS; Department of Biochemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Riyadh, Saudi Arabia. Electronic address: fataya@ksu.edu.sa.
Jazyk: angličtina
Zdroj: International journal of biological macromolecules [Int J Biol Macromol] 2024 Nov; Vol. 279 (Pt 4), pp. 135458. Date of Electronic Publication: 2024 Sep 07.
DOI: 10.1016/j.ijbiomac.2024.135458
Abstrakt: L-asparaginase is a remarkable antineoplastic enzyme used in medicine for the treatment of acute lymphoblastic leukemia (ALL) as well as in food industries. In this work, the L-asparaginase-II gene from Salmonella paratyphi was codon-optimized, cloned, and expressed in E. coli as a His-tag fusion protein. Then, using a two-step chromatographic procedure it was purified to homogeneity as confirmed by SDS-PAGE, which also showed its monomeric molecular weight to be 37 kDa. This recombinant L-asparaginase II from Salmonella paratyphi (recSalA) was optimally active at pH 7.0 and 40 °C temperature. It was highly specific for L-asparagine as a substrate, while its glutaminase activity was low. The specific activity was found to be 197 U/mg and the kinetics elements K m , V max , and k cat were determined to be 21 mM, 28 μM/min, and 39.6 S -1 , respectively. Thermal stability was assessed using a spectrofluorometer and showed T m value of 45 °C. The in-vitro effects of recombinant asparaginase on three different human cancerous cell lines (MCF7, A549 and Hep-2) by MTT assay showed remarkable anti-proliferative activity. Moreover, recSalA exhibited significant morphological changes in cancer cells and IC 50 values ranged from 28 to 45.5 μg/ml for tested cell lines. To investigate the binding mechanism of SalA, both substrates L-asparagine and l-glutamine were docked with the protein and the binding energy was calculated to be -4.2 kcal mol -1 and - 4.4 kcal mol -1 , respectively. In summary, recSalA has significant efficacy as an anticancer agent with potential implications in oncology while its in-vivo validation needs further investigation.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024. Published by Elsevier B.V.)
Databáze: MEDLINE
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