Plasmid construction

To generate the plasmid expressing PfTPx_Gl47‐EGFP, codon optimized tandem oligonucleotides, encoding residues 1–47 of PfTPx_Gl and a triple glycine linker, were inserted upstream of EGFP in the vector pCTG‐EGFP 20. As this protein has multiple in‐frame methionines, the Kozak context of these methionines was retained so as to match the relative Kozak frequencies between them in Plasmodium 21, 22. The plasmid expressing P30‐mCherry‐HDEL was generated by replacing YFP‐HDEL in the ptub‐P30‐YFP‐HDEL vector 23 with mCherry‐HDEL amplified from ptub‐mCherry. Plasmids encoding PfTPx_GlS‐EGFP and PfTPx_GlΔS‐EGFP were generated by replacing the PfTPx_Gl47 region in pCTG‐PfTPx_Gl47‐EGFP with PfTPx_GlS or PfTPx_GlΔS. In the PfTPx_GlΔS‐EGFP mutant, a methionine was included at the translation start site. The plasmid encoding ERS1–10 was generated by replacing YFP‐HDEL in ptub‐P30‐YFP‐HDEL with GFPS1–10 amplified from the pCMV‐mGFP 1‐10 Hyg Amp (Sandia Biotech, Albuquerque, NM, USA) including a C‐terminal ER retention sequence (HDEL). The plasmid encoding CyS1–10 was generated by replacing EGFP in pCTG‐EGFP with GFPS1–10. The plasmid encoding PfTPx_Gl47‐S11 was generated by replacing EGFP in the construct expressing PfTPx_Gl47‐EGFP with GFPS11 amplified from pCMV‐mGFP Cterm S11 Neo Kan (Sandia Biotech). Plasmids encoding the PfTPx_Gl fusion proteins with ACP [ACP_SP‐PfTPx_GlΔS‐EGFP and PfTPx_GlS‐ACP_TP‐EGFP] were generated by overlap extension PCR with vectors pCTG‐PfTPx_Gl47‐EGFP and pCTG‐ACP‐HA using Phusion polymerase (NEB) 24. The plasmid encoding PfTPx_Gl47‐M26A‐EGFP was synthesized using a mismatched forward primer to amplify pCTG‐PfTPx_Gl47‐EGFP with KAPA HiFi DNA polymerase.

To generate the plasmids expressing GFP fusion proteins in Plasmodium, the region encoding the first 47 residues of PfTPx_Gl, including the start codon context of 12 bp and a triple glycine linker, was amplified from the pET28a‐PfTPx_Gl plasmid containing PfTPx_Gl cloned from Plasmodium mRNA in the case of PfTPx_Gl47‐GFP, and synthesized as tandem oligonucleotides in the case of PfTPx_Gl47‐M26A‐GFP. Each of these fragments was inserted upstream of GFP in the pSSPF2 vector 25. Primers used are listed in Table 1.

Primers used in plasmid construction. Restriction sites are underlined and the mismatch regions are italicized. All primer sequences are shown from 5ʹ to 3ʹ