The influence of polyethylene glycol structure on the conjugation of recombi-nant human interferon α2b overproduced using synthetic gene in E.coli

Abstract

Interferon α2b (IFNα2b) is a cytokine which has antiviral, antiproliveration and immunomudulator activities. IFN α2b has been used as a drug for treatment of hepatitis B and C infections. However, the use of this protein is limited, as rapidly cleared by the kidney. Approaches have been made to prevent this fast renal clearance such as increasing the molecular weight of protein using inert compounds like polyethylene glycol (PEG). The aim of this research was to study the influence of PEG structure on the interferon alpha 2b pegylation. rhIFNα2b was obtained from Eschericia coli BL21 containing synthetic gene for rhIFNα2b. rhIFNα2b was pegylated with two different form of PEGs, i.e linear PEG-carbonyldiimidazole (PEG-CI) and branched, trimethyl succinimidyl polyethylene glycol (TMS(PEG)₁₂). PEG-CI was activated and characterized with Dragendorf reagent and infra red spectrophotometry. rhIFNα2b was pegylated using molar ratio 1:100 and 1:300 (protein:PEG). Characterization was performed on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). PEG 2000 was successfully activated with CDI. rhIFNα2b was only successfully conjugated with TMS(PEG)₁₂ resulting in polyform of pegylated proteins with 1, 3, and 5 molecules PEG attached. The structure of PEG molecule influenced the pegylation of interferon. Linear PEG activated with carbonyldiimidazole showed less reactive to interferon than branced PEG.