A lambda gtll genomic library of a deer isolate of M. paratuberculosis was screened with serum from a sheep with Johne's disease. A number of clones were identified, two of which were selected for further study after being found to contain overlapping fragments of the same gene. Antibody elusion studies showed these clones to correspond to a 34kDa protein in M. paratuberculosis. Both clones have been fully sequenced and were found to contain an open reading frame of 1083bp, translating to a protein of 361 amino acids. The sequence also contained a possible Shine Dalgarno sequence, start codon, and appeared to code for a signal peptide of 39 amino acids, and a pro-peptide of 15 amino acids. Although the amino acid sequence showed no great overall homology to any previously described protein in the EMBL database, it did contain a motif (GDSGG) corresponding to the active serine residue in a chymotrypsin-like serine protease, in which His57 and Ser195 are brought together to form the three dimensional active site of the protease. A similar organization can be found in the M. paratuberculosis 34kDa protein (His102 asp133 Ser215). These three sites, however, show a greater homology (over 70%) to those detected in the HtrA proteins of Escherichia coli (His131 Asp161 Ser136). Salmonella typhimurium (His132 Asp162 Ser137), and Brucella abortus (His 152 Asp182 Ser257), than to the sites in chymotrypsin. These HtrA proteins are thought to be serine proteases, and these three homologous sites may form the three dimensional active site of the enzyme. The native 34kDa protein has been affinity-purified using monoclonal antibodies, and the N terminal sequence of the protein matched the deduced amino acid sequence of the two genomic clones. Experiments are underway to determine any proteolytic activity of the native protein.