Fig. 3. PEG conjugation studies of truncated N-terminal His-tagged IFN variants (Tags 1, 3 and 4) and native IFN used as control. Protein to PEG₁₀-mono-sulfone 3 ratios were screened and the conjugation reaction was conducted for 16 h at 20 °C. It was observed that the most effective conjugation at the histidine-tag was achieved when reaction was conducted at pH 5.0 at a protein concentration of 1 mg mL^–1 and 5 eq. of PEG₁₀-mono-sulfone 3. The conversion to the mono-PEGylated species ranged from 29–35% for the three IFN variants, while only 7% PEGylation on native IFN. (A) SDS–PAGE gels (colloidal blue) showing the PEG-conjugation reaction mixtures screened and (B) estimates of PEGylation conversion rates using densitometry analysis for the conjugation conducted with protein concentrations of 1.0 and 2.5 mg mL^–1.