Recombinant Expression of Photo-crosslinkable 26S Proteasome Base Subcomplex.

The 26S proteasome complex is the hub for regulated protein degradation in the cell. It is composed of two biochemically distinct complexes: the 20S core particle with proteolytic active sites in an internal chamber and the 19S regulatory particle, consisting of a lid and base subcomplex. The base contains ubiquitin receptors and an AAA+ (ATPases associated with various cellular activities) motor that unfolds substrates prior to degradation. The base subcomplex can be expressed recombinantly in and reconstituted into functional 26S proteasomes , which allows the introduction of unnatural amino acids with novel functions or other mutations that may not be permissible . Here, we present a method for the introduction of the photo-induced crosslinking amino acid p-benzoyl-L-phenylalanine into the proteasomal base subcomplex. This approach has exciting implications for the study of protein-protein interactions of this complex that mediates the degradation of an incredibly diverse protein pool.