Site-specific cross-linking of functional proteins by transglutamination

Microbial transglutaminase (MTG), an enzyme that works in posttranslational modification of proteins, has been applied to in vitro preparation of a bi-functional fusion protein from separately obtained recombinant proteins. A quite simple strategy in which a specific peptidyl linker recognizable by MTG is fused to the N-terminus of proteins of interest has been verified in the preparation of a bi-functional fusion protein using an antibody variable domain (single chain Fv of anti-hen-egg white lysozyme antibody, scFv) and a fluorescent protein (enhanced yellow fluorescent protein, EYFP). The resultant peptidyl linker-fused proteins were readily cross-linked by MTG to only give the heterodimer (i.e. scFv-EYFP fusion protein), suggesting that the reaction proceeded in highly specific manner. As the fusion protein exhibited sufficient bi-functionality in fluorescence immunoassay (FIA), this work shows for the first time a successful enzymatic preparation of a bi-functional fusion protein through recombinantly incorporated specific peptidyl linkers into target proteins.