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

Infect Immun. 2002 Jun;70(6):3101-10. doi: 10.1128/IAI.70.6.3101-3110.2002.


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.

MeSH terms

  • Animals
  • Antibodies, Protozoan / immunology
  • Antigens, Protozoan / biosynthesis
  • Antigens, Protozoan / genetics
  • Antigens, Protozoan / immunology*
  • Antigens, Protozoan / isolation & purification
  • Chromatography, Affinity / methods
  • Chromatography, Ion Exchange / methods
  • Cysteine
  • Edetic Acid
  • Endotoxins
  • Escherichia coli
  • Fermentation
  • Gene Expression
  • Membrane Proteins / biosynthesis
  • Membrane Proteins / genetics
  • Membrane Proteins / immunology*
  • Membrane Proteins / isolation & purification
  • Nickel
  • Plasmodium falciparum / genetics
  • Plasmodium falciparum / immunology*
  • Protein Folding*
  • Protein Structure, Tertiary
  • Protozoan Proteins / biosynthesis
  • Protozoan Proteins / genetics
  • Protozoan Proteins / immunology*
  • Protozoan Proteins / isolation & purification
  • Rabbits
  • Sarcosine / analogs & derivatives*
  • Sucrose


  • Antibodies, Protozoan
  • Antigens, Protozoan
  • Endotoxins
  • Membrane Proteins
  • Protozoan Proteins
  • apical membrane antigen I, Plasmodium
  • Sucrose
  • sarkosyl
  • Nickel
  • Edetic Acid
  • Cysteine
  • Sarcosine