Protein Expression and PuriWcation 45 (2006) 15–21 www.elsevier.com/locate/yprep 1046-5928/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2005.10.002 Prokaryotic expression, puriWcation, and polyclonal antibody production against a novel drug resistance gene of Leishmania donovani clinical isolate Hema Kothari, Pranav Kumar, Neeloo Singh ¤ Drug Target Discovery and Development Division, Central Drug Research Institute, Lucknow, India Received 1 February 2005, and in revised form 24 September 2005 Available online 26 October 2005 Abstract Diseases produced by protozoan parasites are one of the main causes of morbidity and mortality around the world, aVecting millions of people. Among these, leishmaniasis has become the second most common cause of death and the problem is further complicated by the expansion of parasite resistance to the conventional drugs. The high rate of therapeutic failure thus calls for new rational approaches to develop alternative drugs. Understanding resistance mechanisms may help identify new targets for drug development. So we present here the cloning, expression, puriWcation, and antibody production of a gene implicated in imparting resistance to pentavalent antimony (SbV) in clinical isolates of kala azar with a view to gain insight into the novel mechanism of its drug resistance.  2005 Elsevier Inc. All rights reserved. Keywords: Drug resistance; Leishmania donovani; Clinical isolates; Recombinant protein Leishmania species are intracellular protozoan parasites that cause leishmaniasis with a wide range of clinical symp- toms: cutaneous, mucocutaneous, and visceral. Of the three forms visceral leishmaniasis (VL) 1 also known as kala azar is the most severe form and is fatal if left untreated [1]. The global annual burden of VL is estimated to be about 500,000 and 90% of all the cases occur in India, Nepal, Ban- gladesh, and Brazil. In India, the state of Bihar alone accounts for more than 90% of these [2–4]. For six decades, long parenteral courses of pentavalent antimonial (SbV) drugs, such as sodium stibogluconate and meglumine antimoniate, have been used as the Wrst line of treatment for both visceral and cutaneous leishmaniasis [4– 7]. Design of eVective vaccines against leishmaniasis pre- sents daunting diYculties [8–10] and the problems of treat- ment are exacerbated by the spread of resistance to antimony in India [11–14]. About 60% VL patients in Bihar state of India do not respond to pentavalent antimonials [4,12]. New drugs have become available in recent years for the treatment of VL [15–18]. The Wrst oral drug, miltefosine, showed cure rates of 98% when Wrst introduced but is very toxic and the less toxic liposomal formulation of amphoter- icin B (AmBisome) which has to its disadvantage, its very high cost and thus limited use. Thus in spite of resistance being developed, antimonial drugs still remain the mainstay for treatment of visceral leishmaniasis. To date work done on drug resistance in Leishmania using laboratory mutants have established gene ampliWca- tions and transport mutations as the most prevalent mecha- nisms of resistance [19–23]. To conWrm whether similar resistance mechanisms operate in clinical isolates, genomic DNA was extracted from antimonial responsive and unre- sponsive clinical isolates, and digested. Subsequent gel elec- trophoretic analysis showed the presence of 1.253 kb fragment to be consistently ampliWed in the unresponsive isolates tested and upon transfection conferred antimony resistance to wild-type Leishmania thus conWrming its role * Corresponding author. Fax: +91 0522 2223405. E-mail address: neeloo888@yahoo.com (N. Singh). 1 Abbreviations used: VL, visceral leishmaniasis; IPTG, isopropyl--D- thiogalactopyranoside.