Cloning and expression of human L- Asparaginase and study of its anti growth effect on leukemia cell lines
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1
immunology research center, Tabriz university of medical science, Iran
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2
faculty of advanced medical sciences, Tabriz university of medical sciences, Iran
Objectives: L-Asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartate and ammonia (Cantor et al., 2009). L-Asparaginase has an antineoplastic activity selectively reduces the level of L-asparagine in blood and decreases the proliferation of cancerous cells (Avramis et al., 2006). Consequently, it have been widely used as a therapeutic agent in the treatment of acute lymphoblastic leukemia (Kozak et al., 2002). In the present study, we aim to produce of human l- Asparaginase enzyme and investigate of its antineoplastic effect on leukemia cell line.
Method: Total RNA was extracted from PC3 cell line and converted to cDNA. L- Asparaginase gene was amplified by RT-PCR and
cloned into the PGEM- T vector. The positive clones were digested and finally confirmed by sequencing. The product was
expressed in E. Coli using pET-22b expression vector. Recombinant protein contained hexahistidine tag was purified using nick el nitrilotriacetic acid chromatography. C105 cell line were treated by different concentration of purified enzyme. The cytotoxic effect of enzyme were measured by MTT assay.
Result: The sequence of cloned L- Asparaginase gene consisted of 937 bp showed 100% identity with previously reported
sequence of this gene. Expression of L- Asparaginase gene in E. coli resulted in high levels of protein with molecular weight of 32kDa in SDS-PAGE.The result of MTT assay showed that the C105 cell line is considerably sensitive to deduced protein.
Conclusion: In this study the recombinant L- Asparaginase was expressed and the anti proliferative effect of this product on
leukemia cell line was confirmed. Hence, it would be of interest to employ this recombinant protein as anti lucemic drug.
Acknowledgements
This study was supported by a research grant from Immunology research center, Tabriz University of medical science, Tabriz, Iran.
References
Cantor, J.R., M. Stone, E., Chantranupong, L and Georgiou, G. (2009). The Human Asparaginase-Like Protein 1 hASRGL1is an Ntn Hydrolase with β-aspartyl Peptidase Activity. Biochemistry. 48, 11026–11031.
Avramis, V.I., Tiwari, P.N. (2006). Asparaginase (native ASNase or pegylated ASNase) in the treatment of acute lymphoblastic leukemia. Int J Nanomedicine. 1, 241–254.
Kozak, M., Borek, D., Janowski, R., Jaskolski, M. (2002). Crystallization and preliminary crystallographic studies of five crystal forms of Escherichia coli L-asparaginase II (Asp90Glu mutant). Acta Crystallogr D Biol Crystallogr. 58,130–132.
Keywords:
L- Asparaginase,
antineoplastic effect,
recombinant protein,
cloning,
Expression
Conference:
15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.
Presentation Type:
Abstract
Topic:
Translational immunology and immune intervention
Citation:
Mohammadnejad
L,
Baradaran
B,
Shanehbandi
D,
Pourhassan Moghaddam
M and
Sadreddini
S
(2013). Cloning and expression of human L- Asparaginase and study of its anti growth effect on leukemia cell lines.
Front. Immunol.
Conference Abstract:
15th International Congress of Immunology (ICI).
doi: 10.3389/conf.fimmu.2013.02.00192
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Received:
09 Mar 2013;
Published Online:
22 Aug 2013.
*
Correspondence:
Miss. Leila Mohammadnejad, immunology research center, Tabriz university of medical science, Tabriz, Iran, mohammadnejadl@tbzmed.ac.ir