In this study we describe, the construction of a co-expression vector allowing simultaneous production of Thermoplasma volcanium 20S proteasome alpha- and beta-subunits in Escherichia coli. This heterologous expression system provided high level production of fully active 205 proteasome that can be purified easily by using a conventional two-step chromatographic technique. The recombinant proteasome was purified to homogeneity 12-fold with a specific activity of 26.5 U/mg. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of two unique bands of alpha-(24 kDa) and beta-(21 kDa) subunits which were combined into proteolytically active proteasome complex in vivo when they were co-expressed in E. coli. The predominant peptide hydrolyzing activity was measured with typical chromogenic substrate (Ala-Ala-Phe-pNA) for chymotrypsin-like activity. The sequence analyses of the subunit genes showed that functional domains and residues including catalytic groups are highly conserved as compared to other archaeal proteasomes.