SAGE-Hindawi Access to Research Enzyme Research Volume 2011, Article ID 970983, 6 pages doi:10.4061/2011/970983 Research Article Molecular Cloning and Characterization of P4 Nuclease from Leishmania infantum Safar Farajnia, 1, 2, 3, 4 Leila Rahbarnia, 5 Bahram Maleki zanjani, 1 Mohammad Hossein Alimohammadian, 6 Shahin Abdoli Oskoee, 7 Abbas Beh-pajooh, 3 Nazli Saeedi, 2 and Soheila Montazer Saheb 4 1 Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz 51656-65811, Iran 2 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz 51656-65811, Iran 3 Infectious and Tropical Disease Research Center, Tabriz University of Medical Sciences, Tabriz 51656-65811, Iran 4 Tuberculosis and Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz 51656-65811, Iran 5 Deparetment of Agriculture, Zanjan University, Zanjan, Iran 6 Immunology Department, Pasteur Institute of Iran, Tehran, Iran 7 Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz 51656-65811, Iran Correspondence should be addressed to Safar Farajnia, farajnia@gmail.com and Leila Rahbarnia, le.rahbarnia@gmail.com Received 3 January 2011; Revised 31 March 2011; Accepted 25 April 2011 Academic Editor: Ariel M. Silber Copyright © 2011 Safar Farajnia et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Parasite of the genus Leishmania is reliant on the salvage pathway for recycling of ribonucleotides. A class I nuclease enzyme also known as P4 nuclease is involved in salvage of purines in cutaneous Leishmania species but the relevant enzymes have not been characterized in Leishmania infantum (L. infantum). The aim of this study was to clone and characterize the gene encoding class I nuclease in L. infantum. DNA extracted from L. infantum was used for amplification of P4 nuclease gene (Li-P4) by PCR. The product was cloned, sequenced, and expressed in E. coli for further characterization. Analysis of the sequence of Li-P4 revealed that the gene consists of an ORF of 951 bp. Sequence similarity analysis indicated that Li-P4 has a high homology to relevant enzymes of other kintoplastids with the highest homology (88%) to p1/s1 class I nuclease from L. donovani. Western blotting of antirecombinant Li-P4 with promastigote and amastigote stages of L. infantum showed that this nuclease is present in both stages of parasite with higher expression in amastigote stage. The highly conserved nature of this essential enzyme in Leishmania parasites suggests it as a promising drug target for leishmaniasis. 1. Introduction Protozoan parasites of the genus Leishmania cause a spec- trum of clinical disease, including cutaneous, mucocuta- neous, and visceral leishmaniasis (VL). Approximately 12 million people are infected with this parasite worldwide with 1.5–2 million new cases occurring each year [1]. Leishmania parasites are dimorphic organisms which exist as promastigotes in extracellular stage and in the sandfly midgut, and as amastigote that lives intracellularly in the phagolysosomes of macrophages in the mammalian host cells [2, 3]. Unfortunately, currently available treatment regimens are nonselective drugs with significant toxicity and limited efficacy [4, 5]. On the other hand, efforts aimed at the development of vaccines have only achieved low levels of protection in trials in human subjects [6, 7]. Thus, there is an urgent need to identify novel molecular targets that can be exploited for drug development, vaccine design, or both. Purine salvage pathway seems as an attractive target for drug development against Leishmania. Trypanosomatid protozoa such as Leishmania are purine auxotrophs and are totally dependent upon their hosts to provide purine nucleotides for their survival, growth, and multiplication [8, 9]. It has shown that Leishmania promastigotes possess a unique class I nuclease, [10, 11] that involves the salvage of preformed purines through the hydrolysis of either 3 ′ -nucleotides or nucleic acids [12–16]. An intracellular amastigote-specific protein, P-4, with class I nuclease activity has also been