Recombinant Expression of Difficult-to-Express Parasite Proteins: Strategies for Functional and Structural Characterization and Translational Applications

Recombinant proteins are used to characterize parasite enzymes, membrane proteins, antigens, and protein complexes, but some targets are difficult to recover in a soluble and functional form. Expression can be affected by codon usage, intrinsically disordered or low-complexity regions, transmembrane segments, disulfide-bond formation, post-translational processing, and multidomain organization. This review compares bacterial, yeast, insect-cell, mammalian-cell, Leishmania tarentolae, and cell-free expression systems and examines construct design, codon optimization and harmonization, fusion partners, chaperones, expression regulation, and computational tools. It distinguishes results reported for parasite proteins from recommendations based on general recombinant-protein studies. Experimentally determined Plasmodium falciparum structures illustrate the use of X-ray crystallography, solution NMR, and cryo-electron microscopy; the ribosome example is identified as a parasite-derived complex rather than a recombinant product. Molecular docking, molecular dynamics simulations, and binding-energy calculations are discussed as tools for developing and prioritizing hypotheses, not as substitutes for experimental testing. The review links target features to initial construct and host choices and identifies the protein-quality checks needed before structural or functional conclusions are drawn.