The P6 segment of SERA5 can be readily replaced with a tandem affinity-purification tag. IFA of uncloned drug-resistant parasite lines examined just one cycle of drug selection following transfection with control construct pHH1SERA5chimWT (top row), designed to simply reconstitute the wild-type SERA5 coding sequence, or with pHH1SERA5chimDP6mTAP (middle and bottom row), designed to introduce a mini TAP tag into the SERA5 coding sequence in place of the region encompassed by the P6 region (see Supplementary Fig. S8). Fixed parasites were probed with a polyclonal rabbit anti-SERA5 antibody (red) or with the anti-HA3 mAb 12CA5 (green). A significant proportion of the pHH1SERA5chimDP6mTAP-transfected parasites exhibit an HA3-specific signal, indicating rapid integration of the transfection construct. Scale bar, 5 mm. Examination of uncloned drug-resistant cultures by IFA using anti-HA3 antibodies showed a strong positive signal in up to ~20% of schizonts, which co-localised with the anti-SERA5 signal, indicating efficient integration of the input construct in the predicted manner.

Stallmach R, Kavishwar M, Withers-Martinez C, Hackett F, Collins CR, Howell SA, Yeoh S, Knuepfer E, Atid AJ, Holder AA, Blackman MJ. Plasmodium falciparum SERA5 plays a non-enzymatic role in the malarial asexual blood-stage lifecycle. Mol Microbiol. 2015 Jan 20. [Epub ahead of print]