title: High-level cell-free production of the malarial lactate transporter PfFNT as a basis for crystallization trials and directional transport studies.
abstract: The malaria parasite Plasmodium falciparum relies on the function of channel and transport proteins for the uptake of nutrients and the release of metabolic waste products. Inhibition of vital transport processes is an unexploited means for developing novel antimalarial drugs. The recently discovered plasmodial lactate transporter, PfFNT, represents a promising new drug target since the parasite's energy generation by anaerobic glycolysis depends on the rapid secretion of lactate. Yet, membrane proteins, in particular those of malaria parasites, are notoriously difficult to produce and purify in the native, functional form hampering crystallization and biophysical studies. Here, we show synthesis of milligram quantities of correctly folded PfFNT in a cell-free system. Solubilized PfFNT maintained its oligomeric, largely SDS-resistant quaternary structure and appears suitable for setting up crystallization trials. After reconstitution into proteoliposomes, PfFNT was functional as a transporter for formate, acetate, and lactate as determined by a light-scattering assay. Analysis of the accessibility of a protease cleavage site at the N-terminus revealed an even outside-in orientation of the total proteoliposomal PfFNT population that may be due to membrane curvature restrictions. Contrary to previous studies using heterologous expression in cell systems with oppositely oriented PfFNT, the proteoliposomes eventually allow for biophysical transport studies in the native, physiological direction.
authors.affiliation: Christian-Albrechts-University of Kiel, Department of Medicinal and Pharmaceutical Chemistry, Gutenbergstraße 76, 24118 Kiel, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Holm-Bertelsen
authors.affiliation: Christian-Albrechts-University of Kiel, Department of Medicinal and Pharmaceutical Chemistry, Gutenbergstraße 76, 24118 Kiel, Germany.
authors.forename: Sinja
authors.initials: S
authors.lastname: Bock
authors.affiliation: Christian-Albrechts-University of Kiel, Department of Medicinal and Pharmaceutical Chemistry, Gutenbergstraße 76, 24118 Kiel, Germany.
authors.forename: Folknand
authors.initials: F
authors.lastname: Helmstetter
authors.affiliation: Christian-Albrechts-University of Kiel, Department of Medicinal and Pharmaceutical Chemistry, Gutenbergstraße 76, 24118 Kiel, Germany. Electronic address: ebeitz@pharmazie.uni-kiel.de.
authors.forename: Eric
authors.initials: E
authors.lastname: Beitz
mesh_terms: D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D027501:Monocarboxylic Acid Transporters / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010963:Plasmodium falciparum / Q000235:genetics* / Q000378:metabolism; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D027501:Monocarboxylic Acid Transporters; D015800:Protozoan Proteins
keywords: Cell-free protein production; Directionality; Formate-nitrite transporter; Lactate transport; Plasmodium falciparum