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Plasmodium falciparum AMA-1 protein and uses thereof

a technology of plasmodium falciparum and ama-1, which is applied in the field of purified plasmodium falciparum protein, can solve problems such as problematic prokaryotic expression of ama1 from various species, and achieve the effect of optimal reactivity and elimination of contaminating proteins

Inactive Publication Date: 2006-11-23
LANAR DAVID +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for expressing and purifying a recombinant P. falciparum AMA-1 protein that is properly folded and retains its disulfide bridges, which is important for optimal reactivity for vaccine and screening purposes. The method involves a four-step purification scheme that produces a high-purity AMA-1 protein. The purified protein is found to induce a protective immune response in rabbits and can be used as a vaccine, in diagnostic assays, and for production of antibodies. The invention also provides novel vector constructs for recombinant expression of AMA-1 and host cells transformed with the vector. The purified AMA-1 protein and monoclonal or polyclonal antibodies against AMA-1 can be used for malaria antigen detection or therapy of chronic malaria infection. The vaccine is safe, not painful, and should not result in adverse side effects. The invention also provides a method for fermenting and inducing host cells, and a method for bulk fermentation and expression of AMA-1.

Problems solved by technology

Prokaryotic expression of AMA1 from various species has been problematic, primarily due to the formation of insoluble aggregates presumably due to incorrect folding of the protein.

Method used

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  • Plasmodium falciparum AMA-1 protein and uses thereof
  • Plasmodium falciparum AMA-1 protein and uses thereof
  • Plasmodium falciparum AMA-1 protein and uses thereof

Examples

Experimental program
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Effect test

example 1

[0194] Fermentation of E. coli Origami (DE3) expressing the r-AMA1 / E protein at 10 L and 300 L scale: The synthetic gene cloned in the vector pWRMAL was sequenced and the translation of this gene sequence revealed no amino acid changes from the published 3D7 clone sequence (GenBank™ Accession No U65407.1). Fermentation conditions were developed in a 10 L bioreactor and later scaled-up to a 300 L GMP fermentation. The 10 mL fermentation routinely resulted in 150 gm cell paste while the 300 L fermentation resulted in 4.5 kg cell paste. The final plasmid stability for the GMP fermentation was 36%. Although the use of Origami (DE3) increased the proportion of r-AMA1 / E in the soluble fraction (compared to the conventional BL21 (DE3) strain), protein fractionation experiments showed that a majority of r-AMA1 / E was still localized in the insoluble fraction (data not shown).

example 2

[0195] Extraction of r-AMA1 / E in sarkosyl and its enrichment by Ni+2 affinity chromatography: Aliquots were taken from the GMP cell paste lot and a scalable refolding and purification process was developed. During cell lysis soluble and insoluble forms of r-AMA1 / E were extracted with buffer containing 5% sarkosyl. The r-AMA1 / E constituted ˜1-2% of total cell protein estimated by laser densitometry of a SDS-PAGE run under reduced conditions (FIG. 2A, lane 1). Following the first step of purification over Ni+2 column, r-AMA1 / E was enriched to ˜40% of total protein (FIG. 2A, lane 2). A large fraction of r-AMA / E present in the Ni+2 elution, was aggregated as seen on a non-reduced SDS-PAGE (data not shown).

example 3

[0196] Optimization of the refolding conditions: In order to find the optimal refolding conditions, the Ni+2 elution was subjected to rapid dilution in refolding buffers of varying GSH / GSSG ratios, at pH 8.0, in phosphate buffer. Serial dilutions of these test refolding mixtures were coated on a microtiter plate and ELISA reactivity against the conformation specific, inhibitory, monoclonal antibody 4G2dc1, was used as a measure of folding efficiency; while the reactivity to a monoclonal anti-hexa-histidine antibody was used to confirm equivalent coating efficiency. Ratios of GSH / GSSG tested for refolding included 1 / 0.1 mM, 1 / 0.25 mM, 1 / 1 mM, and 0.1 / 1 mM respectively, while phosphate buffer containing EDTA (pH 8.0) alone was used as a control. The GSH / GSSG ratios of 1 / 0.1 mM and 1 / 0.25 mM were found to be equally efficient, both of which gave 4G2dc1 reactivity about 5 times higher than the phosphate buffer control. As the GSH / GSSG ratio of 1 / 0.25 mM had been previously reported for ...

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Abstract

In this application is described the expression and purification of a recombinant Plasmodium falciparum (3D7) AMA-1 ectodomain. The method of the present invention produces a highly purified protein which retains folding and disulfide bridging of the native molecule. The recombinant AMA-1 is useful as a diagnostic reagent, for use in antibody production, and as a protein for use alone, or as part of, a vaccine to prevent malaria.

Description

[0001] This application claims the benefit of priority under 35 U.S.C. §120 from U.S. application Ser. No. 10 / 105,717 filed on Mar. 25, 2002, still pending which claims priority under 35 U.S.C. §119(e) from U.S. application Ser. No. 60 / 278,616 filed on Mar. 26, 2001, now abandoned.FIELD OF THE INVENTION [0002] The present invention relates to a purified Plasmodium falciparum protein. More particularly, this invention is directed to a composition comprising a portion of the Plasmodium falciparum AMA-1 protein for use as a vaccine. INTRODUCTION [0003]Plasmodium falciparum causes more than three million deaths each year, mostly among children below the age of five (World Health Organization Tropical Disease Research, 1997, TDR Twelfth Program Report, p. 57-76). The spread of multi-drug resistant strains of the parasite has underlined an urgent need for a malaria vaccine. Evidence exists both from animal models and human studies that antibodies to erythrocytic and exo-erythrocytic paras...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07H21/02C07H21/04A61K39/00C07K14/445
CPCC07K14/445A61K39/00A61K39/015A61K2039/55566A61K2039/70Y02A50/30
Inventor LANAR, DAVIDDUTTA, SHEETIJWARE, LISANAIR, LALITHA
Owner LANAR DAVID
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