Purification, characterization, and immunogenicity of the refolded ectodomain of the Plasmodium falciparum apical membrane antigen 1 expressed in Escherichia coli

Abstract

The apical membrane antigen 1 (AMA1) has emerged as a promising vaccine candidate against malaria. Advanced evaluation of its protective efficacy in humans requires the production of highly purified and correctly folded protein. We describe here a process for the expression, fermentation, refolding, and purification of the recombinant ectodomain of AMA1 (amino acids 83(Gly) to 531(Glu)) of Plasmodium falciparum (3D7) produced in Escherichia coli. A synthetic gene containing an E. coli codon bias was cloned into a modified pET32 plasmid, and the recombinant protein was produced by using a redox-modified E. coli strain, Origami (DE3). A purification process was developed that included Sarkosyl extraction followed by affinity purification on a Ni-nitrilotriacetic acid column. The recombinant AMA1 was refolded in the presence of reduced and oxidized glutathione and further purified by using two ion-exchange chromatographic steps. The final product, designated AMA1/E, was homogeneous, monomeric, and >99% pure and had low endotoxin content and low host cell contamination. Analysis of AMA1/E showed that it had the predicted primary sequence, and tertiary structure analysis confirmed its compact disulfide-bonded nature. Rabbit antibodies made to the protein recognized the native parasite AMA1 and inhibited the growth of the P. falciparum homologous 3D7 clone in an in vitro assay. Reduction-sensitive epitopes on AMA1/E were shown to be necessary for the production of inhibitory anti-AMA1 antibodies. AMA1/E was recognized by a conformation-dependent, growth-inhibitory monoclonal antibody, 4G2dc1. The process described here was successfully scaled up to produce AMA1/E protein under GMP conditions, and the product was found to induce highly inhibitory antibodies in rabbits.

FIG. 1.

(A) SDS-PAGE analysis of AMA1/E during purification. The results shown are from a lab-grade purification starting with 20 g of cell paste. Protein analysis was done on a 4 to 12% gradient gel under reduced conditions, and the gel was stained with Coomassie blue. The elutions of DEAE and SP Sepharose columns were concentrated on a 3.5-kDa cutoff Centricon concentrator before being loaded. Lane M, molecular mass markers; lane 1, E. coli lysate loaded on the Ni²⁺ column; lane 2, Ni²⁺ column elution; lane 3, DEAE Sepharose column elution (5 μg of protein); lane 4, SP Sepharose column elution (5 μg of protein; lab-grade AMA1/E product). (B) Western blot analysis for E. coli protein detection (Cygnus HCP detection kit). Lanes 1 to 5, Origami (DE3) bacterial lysate with 4,000, 2,000, 1,000, 500, or 250 ng of protein, respectively; lanes 6 to 11, AMA-1/E product with 1, 10, 100, 500, 1,000, or 2,000 ng of protein per well, respectively.

FIG. 2.

Analytical HPLC profile of the AMA1/E product. The detector output at 215 nm with absorbance units (AU) plotted against time (in minutes) is shown. (A) Gel permeation Shodex Protein KW-803 column elution profile with 10 μg of AMA1/E injected (broken line) and an equal volume of final formulation buffer with no protein injected (solid line); (B) reversed-phase C8 Aquapore RP-300 Å column (7 μm, 30 by 2.1 mm) elution profile with a 4-μg AMA1/E injection (solid line) or with 12 μg of AMA1/E reduced in the presence of 6 M GuHCl and 25 mM DTT and injected (broken line), for which a shift in retention time was observed under the same chromatographic conditions. See Materials and Methods for solvent and gradient information.

FIG. 3.

Relative SDS-PAGE mobility, alkylation analysis, and immune reactivity of AMA1/E. Lanes 1, AMA1/E protein (∼200 ng) in 4 M urea; lanes 2, AMA1/E in 4 M urea treated with iodoacetamide; lanes 3, AMA1/E in 4 M urea reduced with DTT followed by iodoacetamide treatment (see Materials and Methods for reaction details). (A) Proteins separated on a nonreducing 4 to 12% gradient gel and stained with Coomassie blue; (B) Western blot of the gel shown in panel A immunostained with monoclonal antibody 4G2dc1 and developed with HRP-POD substrate; (C) Western blot of the gel shown in panel A immunostained with an immune serum pool collected from western Kenya and developed with HRP-POD substrate.

FIG. 4.

Recognition of the parasite AMA1 with anti-AMA1/E antibodies produced in rabbit. (A) Anti-AMA1/E antibodies (R-1) used for IFA on P. falciparum (3D7) parasites fixed with methanol. Merozoites contained within the late-stage schizonts are shown with bright fluorescence. Magnification, ×1,000. (B) Western blot of P. falciparum 3D7 parasite late schizont proteins extracted with SDS-PAGE loading buffer, separated on a nonreducing gel, Western blotted and immunostained with anti-AMA1/E antibodies, and developed with a chemiluminescent substrate. The top and bottom arrows represent ∼76 and 62 kDa, respectively. Lane 1, postimmune rabbit sera (R-1); lane 2, preimmune serum control.

FIG. 5.

Purified anti-AMA1/E IgG inhibited growth by P. falciparum 3D7 parasite in a GIA, and the inhibition was reversed by the addition of the antigen. Shown are the results of a one-cycle GIA with 0.18, 0.35, or 0.7 mg of purified rabbit IgG ml⁻¹ in suspension culture GIA in 48-well plates. •, anti-AMA1/E IgG; ○, anti-RA-AMA1/E IgG. Antigens (5.3 μg ml⁻¹) were added to anti-AMA1/E IgG concentrations of 0.18 mg ml⁻¹. ▴, RA-AMA1/E antigen added; ▪, AMA1/E antigen added (symbols are offset for clarity). The means ± standard deviations are shown, with the number of experiments for each data point represented within parentheses.

FIG. 6.

Competitive ELISA with individual sera. Serum samples at a 1:1,000 dilution were preincubated with either RA-AMA1/E (RA), AMA1/E (RF), or BSA protein (AL). ELISA was done to assay for anti-AMA1/E-specific antibodies. Shown here are the average OD₄₀₅s at a 1:16,000 dilution for AMA1/E-immunized rabbits (R-1, R-2, and R-3) (clear bars) and RA-AMA1/E-immunized rabbits (R-7 and R-10) (black bars). The standard deviations are represented by lines on top of the bars.