Gene synthesis and site-directed mutagenesis

Previous work demonstrated that full-length Plasmodium VIT was expressed at low levels and was functionally impaired in a yeast heterologous expression system. In contrast, N-terminal truncation versions of the transporter demonstrated increased expression levels and activity and were easily detectable in vacuolar membrane fractions of the same system [21]. Therefore, an N-terminal truncation mutant of PfVIT, designated sPfVIT, was designed for this study. The 711 bp sequence encoding the 237 amino acid, D2-36 N-terminal truncation mutant of the VIT from P. falciparum 3D7 (PlasmoDB gene ID: PF3D7_1223700) was codon-optimized for expression in S. cerevisiae and synthesized using a commercially available service (GenScript, USA). To facilitate detection of expressed protein and future protein purification, an in-frame thrombin cleavage site, myc epitope and His₁₀ affinity tag were engineered into the C-terminal end of the protein to give a final construct of 822 bp (see Additional File 1: Figure S1). BamH1 and Xho1 restriction sites were introduced into the 5′ and 3′-ends of the synthetic DNA, respectively, and the codon optimized sequence was ligated into the multiple cloning site of pESC-Leu expression vector (Agilent, UK) that contained the LEU2 selectable marker. This gave rise to pESC-Leu-sPfVIT that encoded a 273 amino acid construct of molecular mass 30.9 kDa, expression of which was placed under control of the GAL1 promoter. Site- directed mutants of sPfVIT used in this study were engineered using a QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, UK) according to the manufacturer’s protocol with the DNA primers listed in Additional File 2: Table S1, and with pESC-Leu-sPfVIT as template DNA. The fidelity of all constructs was verified by DNA sequence analysis (Macrogen Europe, Amsterdam). Plasmids were propagated in Escherichia coli XL10-Gold Ultracompetent cells (Agilent, UK) using carbenicillin for selection, then purified, quantitated and conserved at – 20 °C until required.