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

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.

Fig. 1

PCR product of the ptr1 gene identified on 1% agarose gel electrophoresis. Lane 1, 100-bp DNA ladder marker; Lane 2, ptr1 PCR product.

Fig. 2

Cells induced with isopropyl-ß-D-thiogalacto-pyranoside separated on a12% SDS-PAGE gel. Lane 1, cell lysate before induction; lane 2, cell lysate 1 h after induction; PTR was not expressed; lane 3, cell lysate 2 h after induction (PTR1 was expressed); lane 4, cell lysate 4 h after induction (PTR1 was expressed) and lane 5, protein marker.

Fig. 3

Dot blot analysis of purified recombinant lizard Leishmania PTR1 as the antigen using an antibody against L. major PTR1 (plate A) and T7 Tag monoclonal antibody (plate B) as the primary antibodies, as detected by using a goat anti- mouse IgG-HRP conjugate. Lanes 1 and 4, antigen (PTR1) and lanes 2 and 3, control (bacterial cell lysate).

Fig. 4.

Western-blotting analysis of purified recombinant lizard Leishmania PTR1 as antigen that was detected by T7 tag monoclonal antibody. Lane 1, control (bacterial cell lysate) and lane 2, purified recombinant lizard Leishmania PTR1.