Abstract
We found that some kind of peptidyl linkers can work as peptidyl tags for microbial transglutaminase (MTG)-mediated protein heterodimerization. The Myc epitope (the amino acid sequence: EQKLISEEL) and the T7 epitope (MASMTGGQQMG) that were attached to the N-terminus of enhanced green fluorescent protein (EGFP) were found to serve Gln-tags (i.e. peptidyl tags that provide reactive Gln residues for MTG-mediated protein cross-linking). A calmodulin binding peptide (KRRWKKNFIAVSAANRFKKISSSGAL), the V5 epitope (GKPIPNPLLGLD-ST) and the Strep-tag II (WSHPQFEK) that were attached to the N-terminus of EGFP were found to be Lys-tags that provide reactive Lys residues for the enzymatic cross-linking. The specific Gln-Lys cross-linkage was formed through Gln- and Lys-tags by MTG and the major products were EGFP heterodimers. The reactivity of peptidyl linkers containing reactive Lys residue decreased when they were tethered to C-terminus of EGFP, indicating that the microenvironment or steric hindrance affects more the substrate recognition of MTG than their amino acid sequences. It was found that the insertion of a specific peptide sequence (FERQHMDS, a part of ribonuclease S-peptide) into a loop of EGFP could facilitate the protein cross-linking reaction. These results suggest that commercial peptidyl tags can be employed as specific cross-linkers for MTG-mediated site-specific protein ligation.
