Recombinant Expression of Photo-crosslinkable 26S Proteasome Base Subcomplex

Abstract

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 S. cerevisiae base subcomplex can be expressed recombinantly in E. coli and reconstituted into functional 26S proteasomes in vitro, which allows the introduction of unnatural amino acids with novel functions or other mutations that may not be permissible in vivo. 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.