Expression and characterization of plasmodium falciparum chloroquine resistance transporter (PfCRT) in pichia pastoris

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Expression and characterization of plasmodium falciparum chloroquine resistance transporter (PfCRT) in pichia pastoris

 

Author: Tan, Weiqi
Title: Expression and characterization of plasmodium falciparum chloroquine resistance transporter (PfCRT) in pichia pastoris
Degree: Ph.D.
Year: 2005
Subject: Hong Kong Polytechnic University -- Dissertations
Antimalarials
Plasmodium falciparum
Chloroquine
Malaria
Department: Dept. of Applied Biology and Chemical Technology
Pages: xix, 220 leaves : ill. ; 30 cm
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b1818107
URI: http://theses.lib.polyu.edu.hk/handle/200/465
Abstract: Malaria is one of the major parasitic diseases, with an annual 300-500 million cases and 1 or 2 million fatalities. 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, 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 expression level was further increased using fed-batch fermentation. Newly overexpressed PfCRT was found in the membrane fractions. It has been purified by Immobilized Metal Affinity chromatography. Yeast microsomes containing PfCRT have been characterized in terms of its ability to transport radiolabelled CQ and regulate pH. It was found that CQ uptake of microsomes containing PfCRTr that was from CQ resistant parasites with K76T or K76I mutation was significantly higher than that of vector control, non-modified PfCRT and sensitive PfCRTs (K76). This PfCRT-mediated CQ transport activity was found to be saturable, dose and time-dependent. The transport activity was ATP-dependent and could be inhibited by a well-known CQ resistance reversing agent verapamil, ionophores (e.g. nigericin and valinomycin), and Na+-H+exchanger (NHE) inhibitors (e.g. amiloride and EIPA). Besides CQ, PfCRT was also found to be specific towards amodiaquine and, to a lesser extent, mefloquine, but not to quinine, quinidine and artemisinin. Both amodiaquine and mefloquine are competitive inhibitors. In addition, the internal pH of resistant PfCRTr-containing microsomes was more acidic than that of the control and sensitive PfCRTs-containing microsomes, suggesting that PfCRT may have a pH regulatory role. Furthermore, purified PfCRT (K76, K76T or K76I) has been reconstituted into proteoliposomes in order to further study the exact function of PfCRT. Similar to the microsome system, it was demonstrated that CQ uptake was higher in K76T or K76I PfCRTr than those in K76 PfCRTs or controls. However, unlike the microsome system, it was found that CQ uptake activity was ATP-independent in proteoliposomes system. Furthermore, 3H-CQ uptake was inhibited by the reversing agent verapamil, nigericin, valinomycin, and NHE inhibitors. This observation supports the notion that PfCRT itself can mediate CQ uptake. Moreover, it was found that K76T or K76I PfCRTr also mediated an acidification to a larger extent than that of K76 PfCRTs. This suggests that CQ uptake and pH regulation may be mediated by PfCRT itself. All in all, my project suggests that PfCRT is responsible for mediating CQ resistance in malaria by virtue of its regulation of CQ transport activity and pH of the food vacuole.

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