title: 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.
authors: Department of Immunology, Walter Reed Army Institute of Research, Forest Glen Annex, Silver Spring, Maryland 20910, USA.
authors: Sheetij
authors: S
authors: Dutta
authors: P V
authors: PV
authors: Lalitha
authors: Lisa A
authors: LA
authors: Ware
authors: Arnoldo
authors: A
authors: Barbosa
authors: J Kathleen
authors: JK
authors: Moch
authors: Meredith A
authors: MA
authors: Vassell
authors: Bader B
authors: BB
authors: Fileta
authors: Svetlana
authors: S
authors: Kitov
authors: Nelly
authors: N
authors: Kolodny
authors: D Gray
authors: DG
authors: Heppner
authors: J David
authors: JD
authors: Haynes
authors: David E
authors: DE
authors: Lanar
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D000953:Antigens, Protozoan / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003545:Cysteine; D004492:Edetic Acid; D004731:Endotoxins; D004926:Escherichia coli; D005285:Fermentation; D015870:Gene Expression; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D009532:Nickel; D010963:Plasmodium falciparum / Q000235:genetics / Q000276:immunology*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D011817:Rabbits; D012521:Sarcosine / Q000031:analogs & derivatives*; D013395:Sucrose
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D004731:Endotoxins; D008565:Membrane Proteins; D015800:Protozoan Proteins; C090077:apical membrane antigen I, Plasmodium; D013395:Sucrose; C025231:sarkosyl; D009532:Nickel; D004492:Edetic Acid; D003545:Cysteine; D012521:Sarcosine