NanoLuc (Nluc), a new smaller and brighter luciferase from deep-sea shrimp ,is evaluated as a reporter in P. falciparum. Parasites engineered to express cytosolic, secreted and exported forms of Nluc have been developed) is targeted to the PV and to the RBC. Diagrams of gene constructs and the IFA images are shown on the left and right respectively. Nluc fused at its N-terminus to (A) the N-terminal region of an exported protein (PEXEL), (B) to a secretion signal peptide (SP) or (C) original (cytosolic). The gene encoding each fusion protein was cloned in the pEF vector. Transfected parasites were analysed by IFA using antibodies to detect Nluc and the PVM marker Exp2. DAPI was used for nuclear staining. Size bar = 5 mm. 113 residues of the exported protein Hyp1 (Plasmodium exported protein), containing the PEXEL cleavage site RLLTE, was fused to Nluc at its N-terminus. PEXEL-Nluc parasites were analyzed by immunofluorescence (IFA) with antibodies for Nluc and Exp2, a PVM marker used to delimit the parasite boundaries. PEXEL-Nluc did not concentrate and colocalize with Exp2 rather occupying the entire RBC cytosolic region that lies beyond the PV, suggesting it is correctly exported.

Azevedo MF, Nie CQ, Elsworth B, Charnaud SC, Sanders PR, Crabb BS, Gilson PR. Plasmodium falciparum Transfected with Ultra Bright NanoLuc Luciferase Offers High Sensitivity Detection for the Screening of Growth and Cellular Trafficking Inhibitors. PLoS One. 2014