Abstract
Mutations in the novel membrane protein Pfcrt were recently found to be essential for chloroquine resistance (CQR) in Plasmodium falciparum, the parasite responsible for most lethal human malaria (Fidock, D. A., Nomura, T., Talley, A. K., Cooper, R. A., Dzekunov, S. M., Ferdig, M. T., Ursos, L. M., Sidhu, A. B., Naude, B., Deitsch, K. W., Su, X. Z., Wootton, J. C., Roepe, P. D., and Wellems, T. E. (2000) Mol. Cell 6, 861-871). Pfcrt is localized to the digestive vacuolar membrane of the intraerythrocytic parasite and may function as a transporter. Study of this putative transport function would be greatly assisted by overexpression in yeast followed by characterization of membrane vesicles. Unfortunately, the very high AT content of malarial genes precludes efficient heterologous expression. Thus, we back-translated Pfcrt to design idealized genes with preferred yeast codons, no long poly(A) sequences, and minimal stem-loop structure. We synthesized a designed gene with a two-step PCR method, fused this to N- and C-terminal sequences to aid membrane insertion and purification, and now report efficient expression of wild type and mutant Pfcrt proteins in the plasma membrane of Saccharomyces cerevisiae and Pichia pastoris yeast. To our knowledge, this is the first successful expression of a full-length malarial parasite integral membrane protein in yeast. Purified membranes and inside-out plasma membrane vesicle preparations were used to analyze wild type versus CQR-conferring mutant Pfcrt function, which may include effects on H(+) transport (Dzekunov, S., Ursos, L. M. B., and Roepe, P. D. (2000) Mol. Biochem. Parasitol. 110, 107-124), and to perfect a rapid purification of biotinylated Pfcrt. These data expand on the role of Pfcrt in conferring CQR and define a productive route for analysis of important P. falciparum transport proteins and membrane associated vaccine candidates.
Highlights
Malaria causes ϳ2.5 million deaths annually, mostly children
Since the technology required for fabricating membrane vesicles of various types and for purifying and reconstituting polytopic integral membrane proteins is well developed for yeast, heterologous expression of Pfcrt in yeast would obviously greatly assist further analysis of Pfcrt transport function
Since neither the native wild type or chloroquine resistance (CQR)-associated Pfcrt sequences nor a wild type sequence optimized at the N-terminal 50 codons is expressed in yeast, but the final synthetic genes are, efficient expression of malarial genes in yeast requires conversion of most, but not necessarily all, “unpreferred” codons
Summary
We synthesized a designed gene with a two-step PCR method, fused this to N- and C-terminal sequences to aid membrane insertion and purification, and report efficient expression of wild type and mutant Pfcrt proteins in the plasma membrane of Saccharomyces cerevisiae and Pichia pastoris yeast. This result recently enticed others to construct genes with optimized yeast codon usage for pfsub (a subtilisinlike protease), pfmsp-1 (a merozoite stage-specific surface protein complex), and the antigen Pfs48/45 [22,23,24] These three studies have yielded some additional success; heterologous expression of P. falciparum genes remains extremely difficult, and no successful overexpression of a polytopic integral membrane protein has yet been reported. Analysis of membrane vesicles from these yeast supports the earlier suggestion [1, 3, 5] that Pfcrt is involved in modulating Hϩ transport
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