Cleavable self-aggregating tags (cSAT) for protein expression and purification.

Rapid protein expression and purification remains a critical technological need, in particular as the number of proteins being identified is exploding. In this chapter, we describe a simple and rapid scheme for expression and purification of recombinant proteins using Escherichia coli, by taking advantage of two self-aggregating peptide fusion tags 18A (EWLKAFYEKVLEKLKELF) and ELK16 (LELELKLKLELELKLK) that can drive target proteins into active protein aggregates in vivo. In practice, a target protein is fused at the N-terminus of the self-cleavable Mxe GyrA intein, which is followed by the 18A or ELK16 tag. The fusion protein is first expressed in the form of active aggregate and then separated by centrifugation upon cell lysis. Subsequently, the DTT-mediated intein self-cleavage reaction releases the target protein into solution. These cleavable self-aggregating tags (cSAT, intein-18A/ELK16) provide a quick and efficient route for the production of proteins with modest purity (around 90% in the case of intein-ELK16). Two application examples are included in the chapter.