A UBIQUITIN-SPECIFIC PROTEASE THAT EFFICIENTLY CLEAVES THE UBIQUITIN-PROLINE BOND

Ubiquitin is a small eukaryotic protein that is synthesized naturally as one of several fusion proteins, which are processed by ubiquitin-sp ecific proteases to release free ubiquitin. The expression of heterolo gous proteins as fusions to ubiquitin in either prokaryotic or eukaryo tic hosts often dramatically enhances their yield, and allows the expo sure of any amino acid following cleavage of ubiquitin. The single exc eption is when proline is the amino acid immediately following ubiquit in; the ubiquitin-proline bond is poorly cleaved by presently studied ubiquitin-specific proteases. We show that the mouse ubiquitin-specifi c protease Unp, and its human homolog Unph, can efficiently cleave the ubiquitin-proline bond in ubiquitin fusion proteins of different size s. N-terminal sequencing of the cleavage products reveals that cleavag e occurs precisely at the ubiquitin-proline junction. The biological s ignificance of this cleavage activity is unclear, as ubiquitin-proline fusions do not occur naturally. However, it may indicate a different catalytic mechanism for these ubiquitin-specific proteases and/or that they can cleave ubiquitin-like proteins. Unp and Unph thus represent versatile ubiquitin-specific proteases for cleaving ubiquitin-fusion p roteins in biotechnology and basic research, regardless of both the am ino acid immediately following ubiquitin, and the size of the fusion p artner.