Site-specific Radiolabeling of His-tagged G3 Variants

Site-specific radiolabeling of histidine tag-containing G3 variants with [[^99mTc]Tc(CO)₃]⁺ was performed as described earlier by Deyev et al.^{22 (link)}. Briefly, the eluate (500 μL) containing ca. 3–5 GBq of [^99mTc]Tc was added to a CRS kit, which was then incubated at 100 °C for 30 min. After incubation, 12 μL from the CRS reaction mixture was added to 40 μg (2.75 nmol) of a G3 variant in 33 μL of PBS. The resulting mixture was incubated for 60 min at 60 °C. Then, a 5000-fold molar excess of histidine (13.7 μmol, 212 μL of 10 mg/mL in PBS) was added to the mixture and further incubated for 15 min at 60 °C. Radiolabeled G3 variants were purified using NAP-5 columns pre-equilibrated and eluted with PBS. Radiochemical yield and purity were measured using radio-iTLC in PBS^{24 (link)}. The radiolabeled DARPins and the reduced- hydrolyzed technetium (RHT) colloid remained at the application point, while [^99mTc]TcO₄⁻, [[^99mTc]Tc(CO)₃]⁺ and its complex with histidine migrated with the solvent front. To determine the presence of RHT, iTLC strips were eluted with pyridine:acetic acid:water (10:3:1.5)^{24 (link)}. In this system, the RHT colloid stayed at the application point, while radiolabeled DARPins, [^99mTc]TcO₄⁻, [[^99mTc]Tc(CO)₃]⁺ and its complex with histidine migrated with the solvent front.
Stability test was performed by incubating the purified radiolabeled proteins with 5000-fold molar excess of histidine in PBS for up to 3 h at room temperature; control samples were incubated in PBS. Samples were analyzed by radio-iTLC analysis in PBS^{22 (link)}.