Purification and initial characterization of Plasmodium falciparum K + channels, PfKch1 and PfKch2 produced in Saccharom
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Microbial Cell Factories Open Access
RESEARCH
Purification and initial characterization of Plasmodium falciparum K+ channels, PfKch1 and PfKch2 produced in Saccharomyces cerevisiae Karen Molbaek1,2, Maria Tejada1, Christina Hoeier Ricke1, Peter Scharff‑Poulsen1, Peter Ellekvist3, Claus Helix‑Nielsen4,5,6, Nirbhay Kumar7, Dan A. Klaerke1* and Per Amstrup Pedersen2*
Abstract Resistance towards known antimalarial drugs poses a significant problem, urging for novel drugs that target vital pro‑ teins in the malaria parasite Plasmodium falciparum. However, recombinant production of malaria proteins is notori‑ ously difficult. To address this, we have investigated two putative K+ channels, PfKch1 and PfKch2, identified in the P. falciparum genome. We show that PfKch1 and PfKch2 and a C-terminally truncated version of PfKch1 ( PfKch11−1094) could indeed be functionally expressed in vivo, since a K+-uptake deficient Saccharomyces cerevisiae strain was complemented by the P. falciparum cDNAs. PfKch11−1094-GFP and GFP-PfKch2 fusion proteins were overexpressed in yeast, purified and reconstituted in lipid bilayers to determine their electrophysiological activity. Single channel conductance amounted to 16 ± 1 pS for PfKch11−1094-GFP and 28 ± 2 pS for GFP-PfKch2. We predicted regulator of K+-conductance (RCK) domains in the C-terminals of both channels, and we accordingly measured channel activity in the presence of Ca2+. Keywords: Malaria, K-channels, Yeast, Recombinant protein Introduction The recent decade has experienced a dramatic decrease in malaria prevalence and morbidity, which is partly due to combined efforts including rapid diagnostics, prompt treatment based on artemisinin combination therapies and the use of insecticide treated nets [1]. However, an estimated 228 million cases still occur annually, leading to around 405,000 deaths a year [2], and the occurrence of resistance towards several artemisinin based combination therapies now calls for the development of new *Correspondence: [email protected]; [email protected] 1 Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg 1870, Denmark 2 Department of Biology, University of Copenhagen, Copenhagen 2100, Denmark Full list of author information is available at the end of the article
drugs [3, 4], preferably aimed at new targets. A recent reverse genetic screen of the murine malaria model P. berghei, using the PlasmoGEM database knock out vector library [5], suggested that a staggering two-thirds of its genome may be essential for normal intra-erythrocytic growth [6]. Bioinformatics analysis of the P. falciparum genome has revealed new potential drug targets such as membrane transporters, channels and pores [7, 8]. A number of membrane transporters from P. falciparum has subsequently been cloned and characterized in Xenopus laevis oocytes [9–13] and some have been shown to be crucial to asexual parasite development. Two genes encoding putative K+ channels PfKch1(Uniprot Q8I5E6) and PfKch2 (Uniprot Q8IKI3) were identified in
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