Integration of PEGylation and refolding for renaturation of recombinant proteins from insoluble aggregates produced in bacteria—Application to a single-chain Fv fragment |
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Authors: | Izumi Kumagai Ryutaro AsanoTakeshi Nakanishi Kentaro HashikamiSho Tanaka Adel BadranHideaki Sanada Mitsuo Umetsu |
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Affiliation: | Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba 6-6-11-607, Aramaki, Aoba-ku, Sendai 980-8579, Japan |
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Abstract: | The conjugation of polyethylene glycol (PEGylation) with downsized compact antibodies is an effective method for overcoming the problem of rapid elimination of the compact antibodies from the body. We integrated site-specific PEGylation with the refolding of a single-chain Fv (scFv) of humanized monoclonal antibody 528 with affinity for the epidermal growth factor receptor, to prepare active PEGylated scFv from insoluble aggregates produced in an Escherichiacoli expression system. The insertion of a cysteine residue at the C-terminus of scFv to serve as the conjugation site for PEG led to the formation of highly multimeric scFv during the refolding process; however, PEGylation after refolding drastically dispersed the multimer into monomeric active scFv fragments. Further, the PEGylation of partially refolded scFv during the refolding process improved the PEGylation efficiency and suppressed the formation of highly multimeric scFv; consequently, monomeric active scFv fragments were obtained directly from the insoluble aggregates in E. coli. We show that in vitro refolding of PEGylated scFv should be useful for improving downstream processing performance in the production of clinically useful small antibodies from insoluble fractions. |
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Keywords: | EDTA, ethylenediaminetetraacetic acid EGFR, epidermal growth factor receptor Fab, antigen binding fragment Fv, variable fragment mPEG, maleimide-PEG scFv, single-chain Fv Mw, molecular weight PEG, polyethylene glycol SDS&ndash PAGE, sodium dodecyl sulfate&ndash polyacrylamide gel electrophoresis VH, heavy chain in Fv VL, light chain in Fv YT, yeast triptone |
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