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.

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