Ginn, C;
Choi, J-W;
Brocchini, S;
(2016)
Disulfide-bridging PEGylation during refolding for the more efficient production of modified proteins.
Biotechnology Journal
, 11
(8)
pp. 1088-1099.
10.1002/biot.201600035.
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Abstract
Proteins that are modified by chemical conjugation require at least two separate purification processes. First the bulk protein is purified, and then after chemical conjugation, a second purification process is required to obtain the modified protein. In an effort to develop new enabling technologies to integrate bioprocessing and protein modification, we describe the use of disulfide-bridging conjugation to conduct PEGylation during protein refolding. Preliminary experiments using a PEG-mono-sulfone reagent with partially unfolded leptin and unfolded RNAse T1 indicated that the cysteine thiols underwent disulfide-bridging conjugation to give the PEGylated proteins. Interferon-β1b (IFN-β1b) was then expressed in E.coli as inclusion bodies and found to undergo disulfide bridging-conjugation during refolding. The PEG-IFN-β1b was isolated by ion-exchange chromatography and displayed in vitro biological activity. In the absence of the PEGylation reagent, IFN-β1b refolding was less efficient and yielded protein aggregates. No PEGylation was observed if the cysteines on IFN-β1b were first modified with iodoacetamide prior to refolding. Our results demonstrate that the simultaneous refolding and disulfide bridging PEGylation of proteins could be a useful strategy in the development of affordable modified protein therapeutics.
| Type: | Article |
|---|---|
| Title: | Disulfide-bridging PEGylation during refolding for the more efficient production of modified proteins |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1002/biot.201600035 |
| Publisher version: | http://doi.org/10.1002/biot.201600035 |
| Language: | English |
| Additional information: | Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. This is the peer reviewed version of the following article: Ginn, C; Choi, J-W; Brocchini, S; (2016) Disulfide-bridging PEGylation during refolding for the more efficient production of modified proteins. Biotechnology Journal , 11 (8) pp. 1088-1099. 10.1002/biot.201600035, which has been published in final form at http://doi.org/10.1002/biot.201600035. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. |
| Keywords: | Science & Technology, Life Sciences & Biomedicine, Biochemical Research Methods, Biotechnology & Applied Microbiology, Biochemistry & Molecular Biology, Disulfide-bridging PEGylation, Process integration, Protein modification, Refolding PEGylation, SITE-SPECIFIC PEGYLATION, RECOMBINANT THERAPEUTIC PROTEINS, HUMAN INTERFERON-BETA, MULTIPLE-SCLEROSIS, INCLUSION-BODIES, BONDS, AGGREGATION, PEG, BIOPHARMACEUTICALS, SOLUBILIZATION |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy > Pharmaceutics |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1503876 |
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