Table 1.

Features of covalent protein–protein coupling methods.

Method	Terminus restriction	Redox dependence	Orthogonal pairs	Scar after coupling	Other features or challenges
Split intein	N-intein at C-terminus, C-intein at N-terminus	Yes, for Cys-dependent split inteins	Yes	0–3 aa	–
Sortase	C-terminal-LPXTGX and N-terminal oligoglycine/alanine	No	S. aureus and S. pyogenes enzymes	Small (7 aa)	Need high conc. of oligoglycine reactant; challenging to reach complete conjugation
Tag/Catcher	Tag or Catcher at N- or C-terminus or internal	No	SpyCatcher, SnoopCatcher	Catcher (≥84 aa) and Tag (12–13 aa) retained	High reaction yield
Electrostatic interaction lock	Should not be restricted	Reducing before reaction; oxidizing after reaction	In principle: several coiled-coil pairings known	Moderate (typically 18 aa)	Disulfides may reverse
Unnatural amino acid coupling	None	No	Yes	1 aa in Ag and 1 aa in virus-like particle	Cost/complexity of expression