The 26S proteasome contains a proteolytic core, 20S proteasome, and its regulatory particle, 19S complex. That regulatory particle contains six ATPases that are involved in unfolding and translocation of substrates to the 20S proteasome's catalytic chamber. We expressed ATPase-encoding genes of the regulatory particle of Saccharomyces cerevisiae and found that some recombinant ATPases can self-assemble into a high-molecular-weight protein complex in Escherichia coli. Purification of the Rpt1Rpt2 hetero-complex and the Rpt4 homo-complex for functional characterization demonstrated their contribution to energy-dependent protein degradation. Our finding, production of a functional subunit of the 19S regulatory particle in bacteria, is a simpler and technically advanced system to functionally characterize individual subunits.