Parasite antigens expressed in Escherichia coli. A refined approach for epidemiological analysis.
A simple method is described to generate carrier-free recombinant antigens following their expression in Escherichia coli. A plasmid, called pMSgt11, has been constructed such that the cleavage site for the protease factor Xa separates the recombinant antigen from an enzymatically active beta-galactosidase. Thus, rapid purification of the active beta-galactosidase recombinant protein, followed by digestion with factor Xa, releases the antigen of interest. The pMSgt11 plasmid is compatible with the phage expression vector, lambda gt11 and the feasibility of applying this system has been demonstrated using malarial recombinant antigens. Inserts from lambda gt11 recombinant Plasmodium falciparum clones have been recloned into the EcoRI site of pMSgt11 and the expressed soluble fusion proteins have been purified from crude extracts using a one step affinity chromatography. After protease digestion, the fusion protein cleavage products were analysed by immunoblot with a panel of different human immune sera. We were able to successfully demonstrate specific antibody titers to the parasite-derived carrier-free antigen, without interference from anti-Escherichia coli-specific antibodies. The general application of this approach to epidemiological analysis is discussed.
Scherf
A
A
Unité de Parasitologie Expérimentale, Institut Pasteur, Paris, France.
Mattei
D
D
Schreiber
M
M
D000818:Animals; D000913:Antibodies, Protozoan / Q000032:analysis; D000953:Antigens, Protozoan / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015951:Factor Xa / Q000494:pharmacology; D006801:Humans; D051379:Mice; D010963:Plasmodium falciparum / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology; D012698:Serologic Tests; D001616:beta-Galactosidase / Q000235:genetics
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; D015951:Factor Xa