Protocol abstract
Nature Protocols 1, - 2241 - 2252 (2006)
Published online: 14 December 2006 | doi:10.1038/nprot.2006.346
Subject Categories: Chemical modification | Isolation, purification and separation | Spectroscopy and structural analysis
PEGylation of native disulfide bonds in proteins
Steve Brocchini1, Sibu Balan1, Antony Godwin1, Ji-Won Choi3, Mire Zloh2 & Sunil Shaunak3
Abstract
PEGylation has turned proteins into important new biopharmaceuticals. The fundamental problems with the existing approaches to PEGylation are inefficient conjugation and the formation of heterogeneous mixtures. This is because poly(ethylene glycol) (PEG) is usually conjugated to nucleophilic amine residues. Our PEGylation protocol solves these problems by exploiting the chemical reactivity of both of the sulfur atoms in the disulfide bond of many biologically relevant proteins. An accessible disulfide bond is mildly reduced to liberate the two cysteine sulfur atoms without disturbing the protein's tertiary structure. Site-specific PEGylation is achieved with a bis-thiol alkylating PEG reagent that sequentially undergoes conjugation to form a three-carbon bridge. The two sulfur atoms are re-linked with PEG selectively conjugated to the bridge. PEGylation of a protein can be completed in 24 h and purification of the PEG-protein conjugate in another 3 h. We have successfully applied this approach to PEGylation of cytokines, enzymes, antibody fragments and peptides, without destroying their tertiary structure or abolishing their biological activity.
- Department of Pharmaceutics, The School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, UK
- Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, UK.
- Faculty of Medicine, Imperial College London, Hammersmith Hospital, Ducane Road, London W12 0NN, UK.
Correspondence to: Steve Brocchini1 e-mail: steve.brocchini@pharmacy.ac.uk
Correspondence to: Sunil Shaunak3 e-mail: s.shaunak@imperial.ac.uk
