Mycobacterium avium subsp. paratuberculosis (Mpt), the causative agent of Johne’s disease (JD), is a global problem in the agricultural industry. It is estimated that 25% of all U.S. dairy herds are JD positive. One obstacle in the management of JD is the lack a sensitive diagnostic test for use during the early stages of infection. Resuscitation promoting factors (Rpf) are proteins that promote the growth of many species of Actinobacteria. If Rpf proteins could enhance the growth of Mpt, the sensitivity of diagnostic fecal culture could be improved, and the impact of JD on the dairy industry would be significantly reduced. The putative rpf translation products from four open reading frames (ORFs) in the genome of Mpt have been designated as Rpf homologues, but their function as true Rpf proteins has not been demonstrated. Bioinformatic and sequence alignment analysis supported the previous identification of four ORFs as rpf homologues in Mpt, and further indicated that each of these homologues contains motifs demonstrated to be critical for Rpf function. I cloned the Mpt homologue of rpfB in E. coli and optimized the conditions necessary for expression. The recovered expression products were tested against dormant Mpt. Although the recombinant protein exhibited effects in E. coli consistent with Rpf expression, dormant Mpt did not respond to recombinant RpfB. This may have been due to the loss of functional conformation during the purification process. Further, RpfB alone may not be sufficient to resuscitate dormant Mpt.