Ion-exchange-immunoaffinity purification of a recombinant baculovirus Plasmodium falciparum apical membrane antigen, PF83/AMA-1.
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.
Narum
D L
DL
Department of Chronic and Infectious Diseases, Medical Biological Laboratory, TNO, Rijswijk, Netherlands.
Welling
G W
GW
Thomas
A W
AW
D000818:Animals; D000953:Antigens, Protozoan / Q000302:isolation & purification*; D016367:Baculoviridae / Q000235:genetics; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007120:Immunochemistry; D008565:Membrane Proteins / Q000302:isolation & purification*; D010963:Plasmodium falciparum / Q000276:immunology*; D016052:Protozoan Vaccines / Q000737:chemistry; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification
D000953:Antigens, Protozoan; D008565:Membrane Proteins; D016052:Protozoan Vaccines; D011994:Recombinant Proteins