The novel insertion sequence IS900 is unique to M. paratuberculosis and has been successfully used as a specific and sensitive DNA probe. IS900 protein expression was studied. As predicted from the nucleotide sequence, a single protein, p43, was produced. This protein was readily over expressed in recombinant E. coli.. A synthetic peptide, from the predicted amino acid sequence, was used to raise a p43 mono-specific antiserum in rabbits. This reagent identified p43 in Western blots of protein extracts from M. paratuberculosis and recombinant E. coli but not extracts of M. avium. The level of p43 is unusually high for the transposase of an IS element. This has important implications for the relationship between IS900 and M. paratuberculosis. p43 is a hydrophilic protein and is potentially antigenic. The protein has been extracted from recombinant bacterial lysate by affinity chromatography to 95% purity. This will provide a defined antigen to test antibody and cell mediated immune response to M. paratuberculosis and is a potential diagnostic reagent.