High yield bacterial expression and purification of active recombinant PA28αβ complex

The PA28 complexes (also termed REG or 11S complexes) are described as activators of the 20S proteasome, a major intracellular protease in eukaryotic cells. They bind to the ends of the barrel-shaped 20S proteasome, and activate its peptidase activities. The interferon γ inducible PA28αβ, made of the two related subunits PA28α and β, is under sustained investigation as it plays important roles in the production by the proteasome of class I antigen peptides. However, in vitro studies of this complex have been impaired by the difficulty of producing large amount of this protein, mainly due to the poor solubility of its β subunit when expressed in Escherichia coli. Here we describe the construction of a bicistronic vector, allowing simultaneous production of functional human PA28α and β subunits in E. coli. Co-expression of the two proteins allows efficient formation of active PA28αβ complexes, that remain soluble and can be easily purified by regular chromatographic procedures.