Assembling proteins in close vicinity to each other provides an opportunity to gain unique function because collaborative and even synergistic functionalities can be expected in an assembled form. There have been a variety of strategies to synthesize functional protein assemblies but site-specific covalent assembly of monomeric protein units without impairing their intrinsic function remains challenging. Herein we report a powerful strategy to design protein assemblies by using microbial transglutaminase (MTG). A serendipitous discovery of self-crosslinking of enhanced green fluorescent protein (EGFP) fused with StrepTag I at the C-terminus revealed that EGFP was assembled through the crosslinking of the Lys (K) residue in the C-terminus of EGFP and the Gln (Q) residue in StrepTag I (AWRHPQFGG). Site-directed mutagenesis of the residues next to the K and Q yielded EGFP assemblies with higher molecular weights. An optimized peptide tag comprised of both K and Q residues (HKRWRHYQRGG) enabled the assembly of different types of proteins of interest (POI) when it was fused to either the N- or C-terminus. The peptide tag that enabled the self-polymerization of the functional POI without a scaffold was designated as a 'PolyTag'.