Integration of PEGylation and refolding for renaturation of recombinant proteins from insoluble aggregates produced in bacteria-Application to a single-chain Fv fragment

Izumi Kumagai, Ryutaro Asano, Takeshi Nakanishi, Kentaro Hashikami, Sho Tanaka, Adel Badran, Hideaki Sanada, Mitsuo Umetsu

研究成果: Article査読

12 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)447-452
ページ数6
ジャーナルJournal of Bioscience and Bioengineering
109
5
DOI
出版ステータスPublished - 2010 5

ASJC Scopus subject areas

  • バイオテクノロジー
  • バイオエンジニアリング
  • 応用微生物学とバイオテクノロジー

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