Ion-exchange-immunoaffinity purification of a recombinant baculovirus Plasmodium falciparum apical membrane antigen, PF83/AMA-1
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D L Narum1, G W Welling, A W Thomas
1Department of Chronic and Infectious Diseases, Medical Biological Laboratory, TNO, Rijswijk, Netherlands.
Abstract:
A two-step purification regime has been developed for a quantitatively minor, putatively transmembrane, M(r) 83,000, apical membrane blood stage vaccine candidate antigen of Plasmodium falciparum (PF83/AMA-1), that has been expressed as a full-length baculovirus recombinant protein, PF83-7G8-1. The first step utilizes a new approach to high-performance ion-exchange chromatography (HPIEC) in which elution conditions are not only defined by charge, but also by hydrophobicity. HPIEC fractionation involves successive sodium chloride gradient anion-exchange elutions (A and B), where a change in the non-ionic detergent polyoxyethylenealkylether C10E5 concentration between elutions A and B (from 0.01% to 0.1% (w/v) respectively), results in a fraction that comprises from 2% to 9% PF83-7G8-1. Subsequent column immunoaffinity purification of this fraction on Q-Sepharose CL 4B-28G2dc1 mAb yields a PF83-7G8-1 preparation that is 56% pure. Rat mAb 28G2dc1 recognizes a C-terminal region that is conserved and cross reactive within the AMA-1 family, thus permitting recombinant and native full-length AMA-1 molecules from other species to be purified for molecular analysis. Immunological and molecular characterisation of the vaccine-related characteristics of purified PF83/AMA-1 are now underway.
Insights
A novel two-step purification method enhances the recovery of Plasmodium falciparum apical membrane antigen-1 (PF83/AMA-1), a key malaria vaccine candidate. This process combines unique ion-exchange chromatography with immunoaffinity purification for improved antigen characterization.
Area of Science:
- Parasitology
- Immunology
- Biochemistry
Background:
- Plasmodium falciparum apical membrane antigen-1 (PF83/AMA-1) is a crucial blood-stage vaccine candidate.
- This antigen is quantitatively minor and putatively transmembrane, posing purification challenges.
- Recombinant full-length PF83/AMA-1 (PF83-7G8-1) has been expressed using baculovirus technology.
Purpose of the Study:
- To develop an effective two-step purification regime for PF83/AMA-1.
- To enable detailed immunological and molecular characterization of the vaccine candidate.
- To facilitate purification of AMA-1 molecules from other species.
Main Methods:
- A novel high-performance ion-exchange chromatography (HPIEC) approach utilizing both charge and hydrophobicity for elution.
- Sequential sodium chloride gradient anion-exchange elutions (A and B) with varying non-ionic detergent concentrations (C10E5).
- Immunoaffinity purification of the HPIEC fraction using Q-Sepharose CL 4B-28G2dc1 mAb.
Main Results:
- The HPIEC step yielded a fraction containing 2-9% PF83-7G8-1.
- Subsequent immunoaffinity purification resulted in a PF83-7G8-1 preparation of 56% purity.
- The rat mAb 28G2dc1 recognizes a conserved, cross-reactive C-terminal region of AMA-1.
Conclusions:
- A robust purification strategy for PF83/AMA-1 has been established.
- The method allows for the purification of recombinant and native AMA-1 molecules for further analysis.
- Ongoing studies will focus on the immunological and molecular characteristics of the purified antigen for vaccine development.