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Title: Functional reconstitution of purified chloroquine resistance membrane transporter expressed in yeast
Authors: Tan, W
Gou, DM
Tai, E
Zhao, YZ
Chow, LMC 
Issue Date: 2006
Source: Archives of biochemistry and biophysics, 2006, v. 452, no. 2, p. 119-128
Abstract: Malaria is one of the major parasitic diseases. Current treatment of malaria is seriously hampered by the emergence of drug resistant cases. A once-effective drug chloroquine (CQ) has been rendered almost useless. The mechanism of CQ resistance is complicated and largely unknown. Recently, a novel transmembrane protein, Plasmodium falciparum chloroquine resistance transporter (PfCRT), has fulfilled all the requirements of being the CQ resistance gene. In order to elucidate the mechanism how PfCRT mediates CQ resistance, we have cloned the cDNA from a CQ sensitive parasite (3D7) and tried to express it in Pichia pastoris (P. pastoris) but with unsuccessful results due to AT-rich sequences in the malaria genome. We have therefore, based on the codon usage in P. pastoris, chemically synthesized a codon-modified pfcrt with an overall 55% AT content. This codon-modified pfcrt has now been successfully expressed in P. pastoris. The expressed PfCRT has been purified with immuno metal affinity chromatography (IMAC) and then reconstituted into proteoliposome. It was found that proteoliposomes have a saturable, concentration and time-dependent CQ transport activity. In addition, we found that proteoliposomes with resistant PfCRTr (K76T or K76I) showed an increased CQ transport activity compared to liposomes with lipid alone, or proteoliposomes reconstituted with sensitive PfCRTs (K76) protein. This activity could be inhibited by nigericin and decreased with the removal of Cl−. This work suggests that PfCRT is mediating CQR in P. falciparum by virtue of its changes in CQ transport activity depending on pH gradient and chloride ion in the food vacuole.
Keywords: PfCRT
Yeast expression system
CQ transport activity
Publisher: Academic Press
Journal: Archives of biochemistry and biophysics 
ISSN: 0003-9861
EISSN: 1096-0384
DOI: 10.1016/
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