Semi-synthesis of the C-terminal region of PfRALP1: an alternative strategy for producing a hard-to-express merozoite protein.

Plasmodium falciparum rhoptry-associated leucine zipper-like protein 1 (PfRALP1) is an indispensable protein involved in merozoite invasion of human red blood cells (RBCs), leading to the deadly malaria disease. PfRALP1 is stored in the rhoptry neck of merozoites and translocated to the moving junction during invasion and is therefore regarded as a moving junction protein. The C-terminal region of the full-length PfRALP1 (PfRALP1-C1) plays a crucial role in binding to an unidentified receptor protein on erythrocytes during merozoite invasion. PfRALP1 also elicits immunogenicity in P. falciparum-infected asymptomatic and semi-immune adults. Successful recombinant expression of PfRALP1-C1 was demonstrated using a wheat germ cell-free extract system. However, obtaining pure recombinant PfRALP1 in other heterologous systems has not yet been explored. We faced significant challenges in obtaining PfRALP1-C1 in pure form in reasonable quantities through overexpression in E. coli. To address this issue, we introduce a semi-synthetic approach to obtain functional PfRALP1-C1. Our findings include an efficient three-segment native chemical ligation of two chemically synthesized peptide segments and a recombinantly expressed polypeptide. The circular dichroism data for the semi-synthetic protein are consistent with the predicted secondary structure, which consists of intrinsically disordered regions and alpha helices. The semi-synthetic PfRALP1-C1 retains the binding epitope and interacts with the unknown RBC receptor, as it competitively inhibits the invasion of RBCs by merozoites. Thus the semi-synthetic method described here offers a promising strategy for producing this difficult-to-express antigenic protein PfRALP1-C1, with potential for large-scale production and downstream biological and therapeutic applications.