The p43 antigen encoded by the positive strand of IS900 has been shown to be expressed in a processed 29kDa form in M. paratuberculosis. Since the initial identification of P43 a number of recombinant forms of the protein have been produced in E. coli. p43 is a highly species specific antigen and the aim of this work was to produce sufficient purified material to evaluate its potential as an immunodiagnostic reagent for M. paratuberculosis in Johne's disease and human Crohn's disease. Initially a good purification of p43 was achieved by DNA-cellulose affinity chromatography. Though the yield was 100-200 *g and purity 90% the method was inapplicable due to degradation of the DNA matrix by residual nuclease activity. Both hydrophobic interaction and ion exchange chromatography were employed as upstream processes to remove this nuclease activity. However p43 was found to bind very tightly to all of these matrices and could only be eluted in denaturing buffers incompatible with DNA-cellulose chromatography. Successful purification has been achieved by engineering the coding sequence and adding 10 histidine residues to the amino, or carboxy, terminus of the protein. The decahistidine tailed forms of p43 have been recovered at 90% purity, with yields of approximately 10 mg/l of bacterial culture, in a single step using immobilized Ni2+ affinity chromatography in the presence of 8 M urea. Downstream processing of this material by preparative SDS PAGE chromatography, using the Bio Rad Prep Cell system, has yielded material >99% pure (by SDS PAGE analysis). We have generated mg quantities of essentially pure p43 which is currently being evaluated for application in immunodiagnosis of ruminant and human M. paratuberculosis infection. This material should be suitable for biological assay including ELISA applications, lymphocyte transformation assays, skin testing in animals and possibly vaccination.