Recombinant expression of the rhoptry protein Clag3.1 as a green fluorescent chimeric protein in Plasmodium falciparum

Abstract

Like all members of Apicomplexa, the motile form of Plasmodium, the merozoite, possesses anelaborate apical complex composed of 3 organelles; micronemes, rhoptries and dense granules.Invasion of erythrocytes by Plasmodium merozoites is a tightly regulated multi-step process thatbegins with the cooperation of proteins released from micronemes and rhoptries to mediateattachment of parasites to the host cell membrane and to establish the parasitophorous vacuole.RhopH complex is a very abundant rhoptry secretion composed of 3 protein members; RhopH1,RhopH2 and RhopH3. RhopH1 is represented by either Clag2, Clag3.1, or Clag9. RhopH2 wasfound to traffic to the rhoptries via its first 24 amino acids. To further address how the othermembers of RhopH complex proteins are trafficked to rhoptries, the present study established anew transgenic Plasmodium falciparum line expressing a large chimeric protein containingalmost ¾ of Clag3.1 fused to the N-terminus of GFP via a linker region under the control ofrhoph2 promoter and its signal peptide encoding sequence. The data showed that this chimerawas expressed efficiently indicating the strength of rhoph2 promoter, however, the chimera wastrapped in the early secretory pathway and did not reach rhoptries. Provided a correct folding ofthis protein, these data could imply that the middle part of Clag3.1 (aa 484-1064) contain someinformation that interfere with the targeting contained in the first 24 amino acids of RhopH2.The C-terminus of Clag3.1 could contain Clag3.1-specific rhoptry targeting information. Basedon literature available in the international databases, this is the first report to successfullyexpress such a large rhoptry chimeric GFP protein.