Characterization of a Schistosoma mansoni cDNA encoding a B-like cyclophilin and its expression in Escherichia coli.
A cDNA encoding a Schistosoma mansoni cyclophilin (SmCyP) has been cloned by polymerase chain reaction amplification using degenerate oligonucleotides based on known conserved cyclophilin (CyP) sequences and by screening an expression cDNA library. The cDNA sequence encodes a 21.5-kDa protein, which shares 59% sequence identity with human CyP B. The SmCyP protein was expressed in Escherichia coli with a hexahistidine affinity tag at its amino terminus and antibodies to the purified (His6)-SmCyP fusion protein were raised in a rabbit. Fractionation of parasite material followed by immunoblot analysis revealed that schistosome CyP is a soluble protein. The N-terminus of the predicted protein contains a hydrophobic region, suggestive of a signal sequence. Accordingly, a recombinant SmCyP protein, lacking the first 23 amino acids was found to share the same gel electrophoretic mobility as the parasite-derived CyP protein, suggesting cleavage of a leader sequence. Hybridization of genomic DNA to a full-length cDNA probe indicates that the SmCyP gene is present as a single copy. Immunohistological experiments in conjunction with confocal scanning laser microscopy and immune electron microscopy show that SmCyP is present in abundance in the adult worm as well as in the schistosomula. The function of CyP in the schistosome is presently unclear, but since its ligand, cyclosporin A, has antischistosomal activity, its function is expected to be a vital one.
Klinkert
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MQ
Istituto di Biologia Cellulare, Consiglio Nazionale delle Ricerche, Rome, Italy.
Bugli
F
F
Engels
B
B
Carrasquillo
E
E
Valle
C
C
Cioli
D
D
D000591:Amino Acid Isomerases / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D003062:Codon; D017124:Conserved Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D018509:DNA, Helminth / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli; D017238:Genes, Helminth; D015801:Helminth Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D019696:Peptidylprolyl Isomerase; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D012550:Schistosoma mansoni / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D003062:Codon; D017931:DNA Primers; D018076:DNA, Complementary; D018509:DNA, Helminth; D015801:Helminth Proteins; D011994:Recombinant Proteins; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase