Gene cloning, overproduction and purification of a functionally active cytoplasmic fatty acid-binding protein (Sj-FABPC) from the human blood fluke Schistosoma japonicum.
We report the gene cloning, molecular characterisation and purification of a 14.7-kDa functionally active recombinant (re) cytoplasmic fatty acid-binding protein (Sj-FABPC) from the Chinese strain of the human bloodfluke Schistosoma japonicum (Sj). As schistosomes are unable to synthesise long chain fatty acids and sterols de novo and must, therefore, take up these lipids from the host, Sj-FABPC is an attractive vaccine and/or drug target. Clone 39 (C39), which contains the entire Sj-FABPC gene, was isolated from a Sj lambda ZAPII cDNA expression library immunoscreened with hyperimmune rabbit serum (HRS) raised against soluble adult Sj proteins. The complete ORF (open reading frame) of Sj-FABPC encodes a protein of 132 amino acids (aa) of 14.7 kDa. The aa sequence of Sj-FABPC exhibits 91% identity to a FABP of S. mansoni (Sm14) and 45% identity to a FABP of Fasciola hepatica (Fh15), putative vaccine candidates for schistosomiasis. Sj-FABPC was subcloned into the QIAexpress vector, pQE-10, and subsequently expressed in Escherichia coli. The re-Sj-FABPC, purified under non-denaturing conditions, was recognized by sera from patients with acute and chronic schistosomiasis japonica. The purified re-Sj-FABPC was also shown to bind to palmitic acid with high affinity. The functional expression of Sj-FABPC will facilitate studies on re-Sj-FABPC to assess its potential as a drug and/or vaccine candidate.
Molecular Parasitology Unit, Queensland Institute of Medical Research, Brisbane, Australia.
M M
MM
Becker
B H
BH
Kalinna
G J
GJ
Waine
D P
DP
McManus
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism*; D017238:Genes, Helminth; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D012549:Schistosoma japonicum / Q000235:genetics* / Q000378:metabolism; D025521:Tumor Suppressor Proteins
D002352:Carrier Proteins; D018076:DNA, Complementary; C107608:FABP7 protein, human; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D009363:Neoplasm Proteins; D025521:Tumor Suppressor Proteins