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Molecular cloning, expression and enzymatic assay of pteridine reductase 1 from Iranian lizard Leishmania

Kazemi, B. and Tohidi, F. and Bandehpour, M. and Yarian, F. (2010) Molecular cloning, expression and enzymatic assay of pteridine reductase 1 from Iranian lizard Leishmania. Iranian Biomedical Journal, 14 (3). pp. 97-102. ISSN 1028852X (ISSN)

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Abstract

Background: Currently, there are no effective vaccines against leishmaniasis, and treatment using pentavalent antimonial drugs is occasionally effective and often toxic for patients. The PTR1 enzyme, which causes antifolate drug resistance in Leishmania parasites encoded by gene pteridine reductase 1 (ptr1). Since Leishmania lacks pteridine and folate metabolism, it cannot synthesize the pteridine moiety from guanine triphosphate. Therefore, it must produce pteridine using PTR1, an essential part of the salvage pathway that reduces oxidized pteridines. Thus, PTR1 is a good drug-target candidate for anti-Leishmania chemotherapy. The aim of this study was the cloning, expression, and enzymatic assay of the ptr1 gene from Iranian lizard Leishmania as a model for further studies on Leishmania. Methods: Promastigote DNA was extracted from the Iranian lizard Leishmania, and the ptr1 gene was amplified using specific primers. The PCR product was cloned, transformed into Escherichia coli strain JM109, and expressed. The recombinant protein (PTR1 enzyme) was then purified and assayed. Results: ptr1 gene was successfully amplified and cloned into expression vector. Recombinant protein (PTR1 enzyme) was purified using affinity chromatography and confirmed by Western-blot and dot blot using anti-Leishmania major PTR1 antibody and anti-T7 tag monoclonal antibody, respectively. The enzymatic assay was confirmed as PTR1 witch performed using 6- biopterin as a substrate and nicotinamide adenine dinucleotide phosphate as a coenzyme. Conclusion: Iranian lizard Leishmania ptr1 was expressed and enzymatic assay was performed successfully.

Item Type: Article
Additional Information: Unmapped bibliographic data: LA - English [Field not mapped to EPrints] J2 - Iran. Biomed. J. [Field not mapped to EPrints] C2 - 21079660 [Field not mapped to EPrints] AD - Cellular and Molecular Biology Research Center, Shahid Beheshti University, Tehran, Iran [Field not mapped to EPrints] AD - Dept. of Parasitology and Mycology, Shahid Beheshti University, Tehran, Iran [Field not mapped to EPrints] AD - Dept. of Parasitology and Mycology, Gorgan University of Medical Sciences, Gorgan, Iran [Field not mapped to EPrints] AD - Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran [Field not mapped to EPrints] DB - Scopus [Field not mapped to EPrints]
Uncontrolled Keywords: Gene expression, Leishmania, Pteridine reductase 1 (PTR1), biopterin, nicotinamide adenine dinucleotide phosphate, pteridine derivative, pteridine reductase 1, unclassified drug, affinity chromatography, article, DNA extraction, dot hybridization, enzyme assay, enzyme purification, gene amplification, Iran, Leishmania, lizard, molecular cloning, nonhuman, nucleotide sequence, polymerase chain reaction, promastigote, protein expression, Western blotting, Animals, Blotting, Western, Cloning, Molecular, DNA, Protozoan, Electrophoresis, Polyacrylamide Gel, Enzyme Assays, Iran, Leishmania, Lizards, Oxidoreductases, Polymerase Chain Reaction, Recombinant Proteins, Reproducibility of Results, Escherichia coli, Leishmania major, lizard Leishmania
Subjects: مقالات نمایه شده محققین دانشگاه در سایت ,Web of Science ,Scopus
Divisions: معاونت تحقیقات و فناوری
Depositing User: GOUMS
Date Deposited: 18 Apr 2015 05:35
Last Modified: 20 Jun 2015 08:43
URI: http://eprints.goums.ac.ir/id/eprint/2203

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