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

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.

[1]  Maria Paola Costi,et al.  Discovery of potent pteridine reductase inhibitors to guide antiparasite drug development , 2008, Proceedings of the National Academy of Sciences.

[2]  P. Kumar,et al.  Degradation of pteridine reductase 1 (PTR1) enzyme during growth phase in the protozoan parasite Leishmania donovani. , 2007, Experimental parasitology.

[3]  A. Fairlamb,et al.  Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate , 2006, Molecular microbiology.

[4]  M. Ouellette,et al.  Unresponsiveness to Glucantime Treatment in Iranian Cutaneous Leishmaniasis due to Drug-Resistant Leishmania tropica Parasites , 2006, PLoS medicine.

[5]  P. Kumar,et al.  Overexpression in Escherichia coli and purification of pteridine reductase (PTR1) from a clinical isolate of Leishmania donovani. , 2004, Protein expression and purification.

[6]  .. B.Kazemi,et al.  Isolation a Lizard Leishmania promastigote from its Natural Host in Iran , 2004 .

[7]  M. Ouellette,et al.  Residues involved in co-factor and substrate binding of the short-chain dehydrogenase/reductase PTR1 producing methotrexate resistance in Leishmania. , 1998, European journal of biochemistry.

[8]  P. Olliaro,et al.  A randomized, placebo-controlled trial of a two-week regimen of aminosidine (paromomycin) ointment for treatment of cutaneous leishmaniasis in Iran. , 1995, The American journal of tropical medicine and hygiene.

[9]  L. Hardy,et al.  PTR1: a reductase mediating salvage of oxidized pteridines and methotrexate resistance in the protozoan parasite Leishmania major. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[10]  I. Feliciello,et al.  A modified alkaline lysis method for the preparation of highly purified plasmid DNA from Escherichia coli. , 1993, Analytical biochemistry.

[11]  M. Ouellette,et al.  A novel antifolate resistance gene on the amplified H circle of Leishmania. , 1992, The EMBO journal.

[12]  J. Beck,et al.  Biopterin conversion to reduced folates by Leishmania donovani promastigotes. , 1991, Molecular and biochemical parasitology.

[13]  M. Ouellette,et al.  Direct and inverted DNA repeats associated with P‐glycoprotein gene amplification in drug resistant Leishmania. , 1991, EMBO Journal.

[14]  R. Miller,et al.  One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[15]  D. Fong,et al.  Beta tubulin gene of the parasitic protozoan Leishmania mexicana. , 1988, Molecular and biochemical parasitology.

[16]  L. Simpson,et al.  Comparison of several lizard Leishmania species and strains in terms of kinetoplast minicircle and maxicircle DNA sequences, nuclear chromosomes, and membrane lipids. , 1988, Molecular and biochemical parasitology.

[17]  F. G. Wallace,et al.  Developmental Stages of Trypanosomatid Flagellates: a New Terminology , 1966, Nature.

[18]  B. Kazemi,et al.  Molecular Cloning and Expression of Iranian Leishmania major Pteridine Reductase 1 , 2008 .

[19]  R. Datta,et al.  Antiparasitic chemotherapy: tinkering with the purine salvage pathway. , 2008, Advances in Experimental Medicine and Biology.

[20]  P. Yates,et al.  Purine and pyrimidine metabolism in Leishmania. , 2008, Advances in experimental medicine and biology.

[21]  B. Smith.  SDS Polyacrylamide Gel Electrophoresis of Proteins. , 1984, Methods in molecular biology.

[22]  B. Smith,et al.  SDS Polyacrylamide Gel Electrophoresis of Proteins. , 1984 .