Schistosoma mansoni: Cloning of a complementary DMA encoding a cytosolic Cu Zn superoxide dismutase and high-yield expression of the enzymatically active gene product in Escherichia coli

Zhi Hong, Philip T. LoVerde, Marie Louise Hammarskjöld, David Rekosh

Research output: Contribution to journalArticlepeer-review

37 Scopus citations

Abstract

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 λgt11 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 (~10%) by incubation with copper and zinc ions.

Original languageEnglish (US)
Pages (from-to)308-322
Number of pages15
JournalExperimental Parasitology
Volume75
Issue number3
DOIs
StatePublished - Nov 1992

Keywords

  • Bacterial expression vector
  • Schistosoma mansoni
  • Superoxide dismutase
  • cDNA clones

ASJC Scopus subject areas

  • Parasitology
  • Immunology
  • Infectious Diseases

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