A suite of polymerase chain reaction-based peptide tagging plasmids for epitope-targeted enzymatic functionalization...

The budding yeast and model eukaryote Saccharomyces cerevisiae has been invaluable for purification and analysis of numerous evolutionarily conserved proteins and multisubunit complexes that cannot be readily reconstituted in Escherichia coli. For many studies, it is desirable to functionalize a particular protein or subunit of a complex with a ligand, fluorophore or other small molecule. Enzyme-catalysed site-specific modification of proteins bearing short peptide tags is a powerful strategy to overcome the limitations associated with traditional nonselective labelling chemistries. Towards this end, we developed a suite of template plasmids for C-terminal tagging with short peptide sequences that can be site-specifically functionalized with high efficiency and selectivity. We have also combined these sequences with the FLAG tag as a handle for purification or immunological detection of the modified protein. We demonstrate the utility of these plasmids by site-specifically labelling the 28-subunit core particle subcomplex of the 26S proteasome with the small molecule fluorophore Cy5. The full set of plasmids has been deposited in the non-profit plasmid repository Addgene (http://www.addgene.org).