Part:BBa_K4247025

mCherry-SnoopCatcher

This part codes for the red fluorescent protein mCherry (part BBa_J06504) fused to SnoopCatcher (part BBa_K4247009) via a linker. The SnoopCatcher enables to form a spontaneous isopeptide bond with any other protein containing the SnoopTag (BBa_K4247008).

Usage & Biology

Since the first successful cloning of the green fluorescent protein GFP of Aequorea victoria in 1992 (Prasher et al. 1992) fluorescent proteins became a widely used tool in many fields of research. Now an array of these proteins exists, with improved variants and a wide colour spectrum. mCherry is a 26.7 kDa protein, and has a short maturation time (15 min), excitation max at 587 nm and emission max at 610 nm (www.fpbase.org). In 2006 its crystal structure owas published (Shu and Remington 2006). Here we provide mCherry with an additional feature, the SnoopCatcher. mCherry-SnoopCatcher has a MW of 40.9 kDa. The Tag-Catcher system is a protein conjugation tool that enables the formation of an irreversible isopeptide bond between these two components. A covalent peptide/protein pair was developed to enable spontaneous isopeptide bond formation between peptide tags. This system was developed based on Gram-positive surface protein, the pilus adhesin RrgA of S. pneumoniae. The D4 domain of this protein is stabilised by an isopeptide forming between a lysine (K742) and an asparagine (N854). This domain was split into a scaffold protein called SnoopCatcher and a 12-residue peptide termed SnoopTag, which can spontaneously form a covalent isopeptide bond upon mixing. The initiation, extension, and release steps use mild conditions, independent of redox state, and therefore should be applicable to a wide range of proteins according to the original paper.