2.4. C-terminal covalent PEGylation of rTM456-LPETG via Sortase A-Mediated Ligation (SML)

rTM₄₅₆-LPETG (14 μM) was mixed with SrtA (2 μM) and NH₂-Gly-Gly-NH-PEG₅₀₀₀-OMe (133 μM) in reaction buffer (20 mM Tris and 150 mM NaCl, pH 8.0). The reaction mixture was then incubated at 37°C for 3 h to obtain the C-terminal PEGylated rTM₄₅₆ (rTM₄₅₆-PEG₅₀₀₀-OMe). The obtained rTM₄₅₆-PEG₅₀₀₀-OMe was purified from the remaining reaction mixture by collecting the pass-through fraction of His SpinTrap column (GE Healthcare). Excess NH₂-Gly-Gly-NH-PEG₅₀₀₀-OMe was then removed by Amicon Ultra Centrifugal Filter (Millipore) with a cutoff molecular weight of 10,000 Da and the rTM₄₅₆-PEG₅₀₀₀-OMe was concentrated. The total amount of pure rTM₄₅₆-PEG₅₀₀₀-OMe was measured by Bradford assay.