title: Expression of functional Plasmodium falciparum enzymes using a wheat germ cell-free system.
abstract: One decade after the sequencing of the Plasmodium falciparum genome, 95% of malaria proteins in the genome cannot be expressed in traditional cell-based expression systems, and the targets of the best new leads for antimalarial drug discovery are either not known or not available in functional form. For a disease that kills up to 1 million people per year, routine expression of recombinant malaria proteins in functional form is needed both for the discovery of new therapeutics and for identification of targets of new drugs. We tested the general utility of cell-free systems for expressing malaria enzymes. Thirteen test enzyme sequences were reverse amplified from total RNA, cloned into a plant-like expression vector, and subjected to cell-free expression in a wheat germ system. Protein electrophoresis and autoradiography confirmed the synthesis of products of expected molecular masses. In rare problematic cases, truncated products were avoided by using synthetic genes carrying wheat codons. Scaled-up production generated 39 to 354 μg of soluble protein per 10 mg of translation lysate. Compared to rare proteins where cell-based systems do produce functional proteins, the cell-free yields are comparable or better. All 13 test products were enzymatically active, without failure. This general path to produce functional malaria proteins should now allow the community to access new tools, such as biologically active protein arrays, and lead to the discovery of new chemical functions, structures, and inhibitors of previously inaccessible malaria gene products.
authors.affiliation: Department of Chemistry, University of Washington, Seattle, Washington, USA.
authors.forename: Devaraja G
authors.initials: DG
authors.lastname: Mudeppa
authors.forename: Pradipsinh K
authors.initials: PK
authors.lastname: Rathod
mesh_terms: D002474:Cell-Free System / Q000378:metabolism*; D015870:Gene Expression; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D014908:Triticum / Q000737:chemistry
chemical_list: D015800:Protozoan Proteins