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.

Fig 1

Autoradiogram of P. falciparum enzymes expressed in a small-batch reaction using the wheat cell-free system. (A) Autoradiogram developed from 14 μg of batch-translated soluble fractions. Inset shows the low-level expression of a GTPCH-radiolabeled band. Lane M, molecular mass marker. (B) Correlation of expected and experimental molecular weights (MW) of newly expressed enzymes.

Fig 2

Visible expression of nonradioactive P. falciparum enzymes in a scaled-up dialysis reaction method. Seven micrograms of soluble fraction of translated lysate was resolved by SDS-PAGE and visualized by Coomassie staining. Based on the densitometry, the respective protein bands are indicated as visible (closed star) and not visible (open star).

Fig 3

Functional analysis of P. falciparum enzymes produced in a cell-free system. Enzyme activities were directly measured in scaled-up expressed translated lysates either by spectrophotometry (A to E and H), scintillation counting (F and G), or visible assays by polyacrylamide gel (K) and TLC (I, J, L, and M). In all cases, GFP-translated lysate served as a control when used at levels corresponding to the largest amount of lysate in experimental functional assays. In the case of the visible assays, the pixel counts for radioactive substrates and products were quantified by ImageJ and converted into moles of activity by using equation 1 (Materials and Methods). Visible assays are shown only for single concentrations (remaining data for the other two concentrations are not shown), but the quantified activity is shown for all three concentrations of translated lysates. In visible assays, substrate-alone reactions are represented as controls (C).

Fig 3

Functional analysis of P. falciparum enzymes produced in a cell-free system. Enzyme activities were directly measured in scaled-up expressed translated lysates either by spectrophotometry (A to E and H), scintillation counting (F and G), or visible assays by polyacrylamide gel (K) and TLC (I, J, L, and M). In all cases, GFP-translated lysate served as a control when used at levels corresponding to the largest amount of lysate in experimental functional assays. In the case of the visible assays, the pixel counts for radioactive substrates and products were quantified by ImageJ and converted into moles of activity by using equation 1 (Materials and Methods). Visible assays are shown only for single concentrations (remaining data for the other two concentrations are not shown), but the quantified activity is shown for all three concentrations of translated lysates. In visible assays, substrate-alone reactions are represented as controls (C).

Fig 4

Optimized codons minimize the truncated products and improve expression levels in a wheat cell-free system. (A) Autoradiogram of native (N) and optimized (O) codons. (B) Protein quantitation of native and optimized codons translated in the dialysis reaction method.