The ubiquitin–proteasome system (UPS) targets selected proteins for degradation by the 26S proteasome. The initial steps in this pathway generate proteins that are covalently tagged with a polyubiquitin chain that is then recognized by ubiquitin receptors of the 26S proteasome. This is a large complex composed of a 20S catalytic core particle and two 19S regulatory particles (Kok et al., 1993) that catalyse the final step in the pathway. While the 20S particle is composed of a catalytic chamber for protein degradation, collectively the proteins that comprise the 19S particle perform several proteasomal functions that include recognition of ubiquitylated substrates, cleavage of the polyubiquitin chain for ubiquitin recycling, control of access to the 20S proteolytic chamber, and substrate unfolding and subsequent translocation into the 20S core particle for degradation (Boehringer et al., 2012). Mammalian proteasomes are associated with three DUBs: USP14, UCHL5 (UCH37) and Rpn11 (POH1). UCHL5 and USP14 reside on the regulatory particle and remove ubiquitin from the substrate before substrate degradation whereas Rpn11's activity is delayed until the proteasome is committed to degrading the substrate (Lee et al., 2010). The DUB activity of USP14 is known to be activated by proteasomes. To fully understand the function and regulation of the proteasome complex, an important step is to elucidate its subunit composition and posttranslational modifications. Toward this goal, Wang et al. (2007) have developed an affinity purification strategy using a derivative of the HB tag for rapid isolation of the human 26S proteasome complex for subsequent proteomic analysis. The purification of the complex is achieved from a stable HEK293 cell line expressing a HB-tagged (6His/Biotin-tagged) proteasome subunit (hRpn11) and by high-affinity streptavidin binding with TEV cleavage elution. The ‘Biotin-tag' consists of a Biotin Acceptor Peptide sequence recognised and biotinylated by the enzyme BirA.
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