Molecular cloning, expression analysis and iron metal cofactor characterisation of a superoxide dismutase from Toxoplasma gondii

A genomic region of 12kb encompassing the gene encoding the superoxide dism utase (SOD) of Toxoplasma gondii has been cloned. The gene contains four ex ons of 121, 42, 381 and 59 bp which are separated by three introns of 321, 202 and 577 by, respectively. The open leading frame can be translated into a protein of 201 amino acids with a molecular mass of 22.6 kDa, Alignment indicated that it is a FeSOD, a type only found in bacterial protozoa and c hloroplast of higher plants. Recombinant SOD was expressed in a Escherichia coli double mutant lacking both MnFeSOD and FeSODs. The presence of iron a s metal cofactor was confirmed by measurements of iron by absorption mass s pectrometry and electron paramagnetic resonance studies. Semi-quantitative reverse transcribed polymerase chain reaction experiments showed a similar amount of SOD transcripts in two developmental stages of T. gondii. Antibod ies raised against the purified recombinant protein detected SOD protein in both bradyzoite and tachyzoite forms suggesting this SOD might be essentia l for the intracellular growth of both developmental stages. Southern blot analysis indicated that SOD occured as a single copy gene in T. gondii geno me. (C) 2000 Elsevier Science B.V. All rights reserved.