Effect of codon optimization on expression levels of a functionally folded malaria vaccine candidate in prokaryotic and eukaryotic expression systems

Abstract

We have produced two synthetic genes that code for the F2 domain located within region II of the 175-kDa Plasmodium falciparum erythrocyte binding antigen (EBA-175) to determine the effects of codon alteration on protein expression in homologous and heterologous host systems. EBA-175 plays a key role in the process of merozoite invasion into erythrocytes through a specific receptor-ligand interaction. The F2 domain of EBA-175 is the ligand that binds to the glycophorin A receptor on human erythrocytes and is therefore a target of vaccine development efforts. We designed synthetic genes based on P. falciparum, Escherichia coli, and Pichia codon usage and expressed recombinant F2 in E. coli and Pichia pastoris. Compared to the expression of the native F2 sequence, conversion to prokaryote (E. coli)- or eukaryote (Pichia)-based codon usage dramatically improved the levels of recombinant protein expression in both E. coli and P. pastoris. The majority of the protein expressed in E. coli, however, was produced as inclusion bodies. The protein expressed in P. pastoris, on the other hand, was expressed as a secreted, soluble protein. The P. pastoris-produced protein was superior to that produced in E. coli based on its ability to bind to red blood cells. Consistent with these observations, the antibodies generated against the Pichia-produced protein prevented the binding of recombinant EBA to red blood cells. These antibodies recognize EBA-175 present on merozoites as well as in sporozoites by immunofluorescence. Our results suggest that the Pichia-based EBA-F2 vaccine construct has further potential to be developed for clinical use.

FIG. 1.

Expression of recombinant F2 in E. coli. (A) Recognition of pQE60-Pf-F2 (native codons) and AKI-eF2 (E. coli codons) by a conformational MAb, MAb RII.10, in a Western blot. Lanes 1 and 2, uninduced and induced pQE60-Pf-F2, respectively; lanes 3 and 4, uninduced and induced AKI-eF2, respectively. Monomeric F2 is indicated by the arrows. (B) Coomassie blue-stained polyacrylamide gel comparing the reduced (lane 1) and nonreduced (lane 2) Ni-NTA-purified AKI-eF2, revealing that the protein tends to form aggregates. (C) Western blot analysis revealing that only a small proportion of the monomeric form of AKI-eF2 is recognized by a conformational MAb (lane 1) compared to the polyclonal anti-EBA antibodies (lane 2).

FIG. 2.

Expression of recombinant F2 in P. pastoris. Western blot analysis revealed that Pf-F2 (native codons) did not show any detectable expression (lane 1). eF2 (E. coli codons) (lane 2) and yF2 (Pichia codons) (lane 3) expressed protein recognized by a conformational MAb.

FIG. 3.

Expression of yF2 (Pichia codon-optimized protein) in P. pastoris. (A) yF2 was detectable in reduced (lane 1) and nonreduced (lane 2) culture supernatants upon Coomassie blue staining. (B) Coomassie blue staining of yF2 purified over Ni-NTA, showing the presence of the monomeric form of the protein (lane 1), which had a reduced mobility following reduction (lane 2). (C) Nonreduced yF2 was recognized to an equal extent by a conformational MAb (lane 1) and polyclonal anti-yF2 antiserum (lane 3). Reduced yF2 was recognized by the polyclonal antibodies (lane 4) but was not recognized by the MAb (lane 2).

FIG. 4.

Analysis of erythrocyte binding capacity of yF2. (A) yF2 (lane 2) was incubated serially with fresh erythrocytes, and the samples were analyzed on a Coomassie blue-stained gel. Coomassie blue staining revealed depletion of yF2 in the supernatant following incubation with normal human erythrocytes. After two serial incubations, there was complete depletion of antigen (lanes 3 and 4). Lane 5, normal erythrocytes incubated with medium alone. Removal of sialic acids upon neuraminidase treatment abolished the ability of yF2 to bind to erythrocytes (lane 1). (B) The erythrocytes described for panel A were washed and eluted with salt to confirm the specificity. In a Western blot analysis, no antigen was detected in the eluted fraction of the neuraminadase-treated erythrocytes (lane 1). F2 was detected in the eluate of normal erythrocytes after the first round of incubation with yF2 (lane 2). No protein was eluted from the cells after the third passage of unbound supernatant (lane 3), confirming that all of the antigen was depleted after two serial incubations with fresh erythrocytes.

FIG. 5.

ELISA IgG titers in sera of mice immunized with reduced and nonreduced forms of F2. (A) Sera from mice immunized with reduced (○) or nonreduced (•) AKI-eF2 had better reactivity to the reduced form of EBA-RII. Sera from mice immunized with yF2 (▪) reacted poorly to the reduced plate antigen. (B) In contrast, sera from mice immunized with yF2 (▪) had high antibody titers to nonreduced EBA-RII. Sera from mice immunized with AKI-eF2 (reduced ○ and nonreduced •) did not recognize the nonreduced plate antigen well. Normal mouse serum (▴) did not react to either reduced or nonreduced plate antigen. O.D., optical density.

FIG. 6.

Functional characterization of anti-F2 antibodies. Sera from mice immunized with yF2 (▪) inhibited the binding of biotinylated glycophorin A to RII, while sera from mice immunized with reduced (○) and nonreduced (•) AKI-eF2 failed to inhibit this binding. Normal mouse serum (▴) served as a control.

FIG. 7.

(A) Mice immunized with yF2 showed a punctate fluorescence pattern on the apical end of schizonts. (B) Sporozoites also demonstrated a punctate recognition along the entire parasite.

FIG. 8.

Anti-yF2 antibodies recognize EBA-175 from culture supernatants from homologous (3D7) (lane 1) as well as heterologous (FVO [lane 2] and ItG [lane 3]) strains of P. falciparum. Lane 4, control lane with complete medium probed with anti-yF2 antibodies.