Sequence, high level expression and purification of a recombinant 21 kDa protein of Cowdria raminantium from Escherichia coli

Abstract

One goal of our project is to provide a convenient, inexpensive and pure source of protein for testing in diagnostic assays and vaccines against heartwater. The DNA insert from recombinant E. coli colony F5.2, expressing an immunodominant Cowdria ruminantium protein, was sequenced. The DNA was AT rich (74 %), hybridized to all isolates of C. ruminantium tested and not to bovine or Anaplasma marginale DNA, and contained two long open reading frames (ORFs) of 627 and 831 base pairs. The ORFs were of enriched GC content compared to the overall base composition and both ORFs potentially encoded proteins containing an N-terminal signal peptide. Deletion experiments followed by expression assays suggested that the 627 bp ORF encoded the immunodominant C. ruminantium protein recognized by infection sera. The ORF for this mature polypeptide was amplified using the polymerase chain reaction and inserted into a high level expression vector where it was expressed as a fusion protein. The attached 9 amino acid fusion peptide enabled rapid purification of the recombinant C. ruminantium protein.