Expression and characterisation of Plasmodium falciparum acidic basic repeat antigen expressed in Escherichia coli.
The acidic basic repeat antigen (ABRA) of Plasmodium falciparum has been localised on the merozoite surface and in the parasitophorous vacuole. It is one of the antigens enriched in the clusters of merozoites formed with growth inhibitory immune serum and possesses chymotrypsin-like activity. Chymostatin, an inhibitor of chymotrypsin, inhibits malaria invasion as well as autoproteolysis of ABRA. Based on these characteristics of ABRA, it seems important for invasion and should be investigated as a target for vaccine and drug design. For the functional characterisation of this protein, the full-length mature ABRA protein and its fragments with/without the putative protease active site were cloned, expressed and purified from Escherichia coli. The polyclonal serum raised against recombinant ABRA fragment recognised a parasite protein with a mobility of 101 kDa in an immunoblot assay and showed immunofluorescence activity with a schizont-rich preparation of P. falciparum. Using a partially purified fragment containing the putative active site and fluorogenic and chromogenic substrates, we established that the protease activity of ABRA resides in the N-terminal portion of the protein and the highly charged C-terminal part of the protein is not required for this activity. The protease activity of ABRA was inhibited with serine protease inhibitors like chymostatin and phenyl methyl sulfonyl fluoride (PMSF) whereas leupeptin was not able to inhibit this enzyme activity. These results clearly indicated that ABRA is a protease with chymotrypsin-like specificity. This is the first report describing the expression and characterisation of recombinant ABRA protein.
Kushwaha
A
A
International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.
Rao
P P
PP
Duttu
V S
VS
Malhotra
P
P
Chauhan
V S
VS
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002918:Chymotrypsin / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015951:Factor Xa / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D010963:Plasmodium falciparum / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000953:Antigens, Protozoan; D017931:DNA Primers; D010446:Peptide Fragments; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D002918:Chymotrypsin; D015951:Factor Xa