Cloning of a cDNA encoding SjIrV1, a Schistosoma japonicum calcium-binding protein similar to calnexin, and expression of the recombinant protein in Escherichia coli.
Membrane-associated proteins were isolated from adult Philippine strain Schistosoma japonicum by partitioning into the detergent phase of Triton X-114. A rabbit polyclonal antiserum raised against these proteins was used to screen an S. japonicum expression cDNA library. Positive clones were identified which encoded the species orthologue of SmIrV1, a Schistosoma mansoni protein which was initially identified by screening with sera from mice protectively vaccinated with irradiated cercariae [Hawn et al., J. Biol. Chem. 268 (1993) 7692-7698]. The S. japonicum molecule, which we term SjIrV1, is 83% identical to SmIrV1 at the predicted amino acid level and is a member of the calreticulin family of non-EF-hand, calcium-binding proteins. The Chinese strain S. japonicum orthologue of SjIrV1 was obtained by screening with the radiolabelled insert of the Philippine strain clone. Northern blot analysis revealed a single message of around 2.4 kb and gave no indication of alternative splicing. Southern blot analysis gave a simple pattern, indicating a single-copy gene, and showed a single restriction fragment length polymorphism between the genomes of Chinese and Philippine strains of S. japonicum. Recombinant, full-length SjIrV1 was expressed with a hexahistidine tag in Escherichia coli and the recombinant protein isolated by nickel-chelate chromatography. Recombinant SjIrV1 was shown to exhibit calcium-dependent, differential electrophoretic migration and to bind ruthenium red in the absence but not in the presence of calcium ions. The presence of conserved Ca(2+)-binding motifs predicted from the primary sequence, together with the Ca(2+)-dependent electrophoretic mobility of recombinant SjIrV1, confirmed that SjIrV1 was a functional calcium-binding protein.
Hooker
C W
CW
Molecular Parasitology Unit, Queensland Institute of Medical Research, Royal Brisbane Hospital, Australia.
Brindley
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D037281:Calnexin; D037282:Calreticulin; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000096:biosynthesis; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D017238:Genes, Helminth; D008969:Molecular Sequence Data; D012261:Ribonucleoproteins / Q000235:genetics; D012549:Schistosoma japonicum / Q000235:genetics*; D012554:Schistosomiasis japonica / Q000517:prevention & control; D016415:Sequence Alignment; D014612:Vaccines / Q000235:genetics