Pet28b

A fragment of the P. falciparum HRP2 gene (109–916 bp) lacking the N-terminal signal peptide was amplified by PCR from plasmid MRA-67 (ATCC/MR4) and cloned into pET28b (Novagen) vectors containing ECFP and EYFP using standard restriction and ligation techniques. To generate sensors based on truncated PfHRP2, a forward oligonucleotide primer was designed to anneal to a repeated PfHRP2 sequence motif identified using MEME (48 (link)) and was used with a fixed reverse primer to PCR amplify fragments of varying sizes using the PfHRP2 gene as a template. These were separated by agarose gel electrophoresis, and then cloned into ECFP and EYFP-containing vectors as above. Fragments mapped to full-length PfHRP2, except for some minor in-frame insertion/deletion mutations in the histidine-rich repeats. For CH₁₈Y, the oligonucleotide encoding the heme-binding motif (HHAHHAADA)₂ was generated in a Klenow reaction and cloned as above. For expression in P. falciparum, coding sequences from pET28b-based vectors were PCR amplified and cloned using the Gibson Assembly Master Mix (New England Biolabs) to replace the ENR-GFP fusion protein in plasmid MRA-846 (ATCC/MR4).