Schistosoma mansoni: cloning of a complementary DNA encoding a cytosolic Cu/Zn superoxide dismutase and high-yield expression of the enzymatically active gene product in Escherichia coli.
We recently purified a 16-kDa cytosolic Cu/Zn superoxide dismutase (CT Cu/Zn-SOD) from Schistosoma mansoni, a human parasite. Three peptide sequences were obtained, one from the unblocked N-terminal and two from internal peptides which were generated by digestions with trypsin and cyanogen bromide. These sequences were aligned to the corresponding sequences of 19 cytosolic Cu/Zn-SODs from various species. Degenerate oligonucleotides were then designed according to the sequence and the position of each peptide. The oligonucleotides were used to amplify a complete cDNA using the polymerase chain reaction with either adult schistosome total RNA or a cercariae lambda gt11 phage cDNA library as the template. The protein encoded by the cDNA has 153 amino acids with a calculated molecular weight of 15,693. It also has 60-65% homology to 19 cytosolic Cu/Zn-SOD from various species. All of the copper/zinc binding sites and SOD activity sites are conserved. Computer analysis predicts that the Cu/Zn-SOD has a pI value of 6.6, which is very close to the experimental results of IEF analysis (6.0 and 6.3). The entire coding sequence from the cDNA was cloned into a bacterial alkaline phosphatase cytosolic expression vector and a large amount of soluble product was expressed and purified to homogeneity. We compared the bacterially expressed Cu/Zn-SOD with the native enzyme derived from schistosomes and found that they are identical by the following criteria: (1) They focus at the same positions on IEF gels; (2) they form dimers in solution as measured by gel filtration; (3) they have the same unblocked N-terminal sequence; (4) they both are enzymatically active with comparable specific activities. The specific activity of the bacterially derived enzyme was increased somewhat (approximately 10%) by incubation with copper and zinc ions.
Hong
Z
Z
Department of Biochemistry, State University of New York, Buffalo 14214.
LoVerde
P T
PT
Hammarskjöld
M L
ML
Rekosh
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D017238:Genes, Helminth / Q000235:genetics*; D015801:Helminth Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012550:Schistosoma mansoni / Q000201:enzymology* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013482:Superoxide Dismutase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014158:Transcription, Genetic
D015801:Helminth Proteins; D011994:Recombinant Proteins; D013482:Superoxide Dismutase