Integration of PEGylation and refolding for renaturation of recombinant proteins from insoluble aggregates produced in bacteria : Application to a single-chain Fv fragment(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
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概要
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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 Escherichia coli 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.
著者
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NAKANISHI Takeshi
Department of Applied Physics and DIMES, Delft University of Technology Lorentzweg 1
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Sanada Hideaki
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University
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Sanada Hideaki
Department Of Applied And Bioapplied Chemistry Graduate School Of Engineering Osaka City University
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Badran Adel
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University
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Kumagai Izumi
Department of Biomolecular Engineering, Tohoku University
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TANAKA Sho
Department of Physics, Kyoto University
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Nakanishi Takeshi
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University
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asano ryutaro
Dept Biomol Eng, Grad Sch Eng, Tohoku Univ.
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Asano Ryutaro
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University
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Hashikami Kentaro
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University
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Umetsu Mitsuo
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University
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Nakanishi Takeshi
Department Of Applied And Bioapplied Chemistry Graduate School Of Engineering Osaka City University
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Hashikami Kentaro
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University
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Kumagai Izumi
Department Of Biochemistry And Engineering Graduate School Of Engineering Tohoku University
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Kumagai I
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University
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Asano Ryutaro
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University
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Umetsu Mitsuo
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University
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Umetsu Mitsuo
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University:center For I
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Tanaka Sho
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University
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Naka Takashi
Innovative Materials Engineering Laboratory National Institute For Material Science
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Umetsu Mitsuo
Dept Biomol Eng Grad Sch Eng Tohoku Univ.
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Kumagai Izumi
Dept Biomol Eng Grad Sch Eng Tohoku Univ.
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