Hanbang Zhang working the Roepe laboratory succeeded in a brute force resolution to this dilemma. Noting the success of earlier gene design that allowed MSP-1 expression in Escherichia coli [85] , Zhang back-translated the PfCRT amino-acid sequence using preferred yeast codons, then designed a set of overlapping 40-mer oligonucleotides that encoded both strands of the theoretically optimized sequence, and constructed an entirely synthetic gene via nested PCR methods. This enabled the expression of high levels of PfCRT protein in either Saccharomyces cerevisiae or Pichia pastoris, in either a constituitive or inducible fashion, respectively [72] . Analysis of ion transport for plasma membrane inside-out vesicles produced from these strains provided further evidence that PfCRT protein plays a role in ion transport [72] , and purified membranes along with equilibrium binding studies, using 3H-CQ, provided the first direct evidence that PfCRT protein indeed binds CQ [73] . The latter is a central prediction for nearly all molecular hypotheses for PfCRT. Although not often cited, the same paper also provided the first direct molecular evidence that PfCRT likely transports CQ, via flow dialysis experiments using inside-out yeast plasma membrane vesicle with or without PfCRT protein [73] . The simplest model consistent with these results is that CQ transport by PfCRT is facilitative downhill diffusion at a rather low turnover. Different thermodynamic models for CQ transport (e.g., PfCRT as a CQ exchanger or active transporter) have also subsequently been proposed based on different approaches that measure 3H-CQ flux for live parasites [86, 87] . Distinction between these models will require additional direct transport experiments at much higher kinetic resolution.