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

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)447-452
Number of pages6
JournalJournal of Bioscience and Bioengineering
Volume109
Issue number5
DOIs
Publication statusPublished - 2010 May

Keywords

  • In vitro refolding
  • PEGylation
  • Recombinant antibody
  • Therapeutic protein
  • scFv

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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