Functional construction of the anti-mucin core protein (MUC1) antibody MUSE11 variable regions in a bacterial expression system

Ryutaro Asano, Shin Ichi Takemura, Kouhei Tsumoto, Naoki Sakurai, Atsushi Teramae, Shinji Ebara, Yu Katayose, Masao Shinoda, Masanori Suzuki, Kohzoh Imai, Seiki Matsuno, Toshio Kudo, Izumi Kumagai

Research output: Contribution to journalArticlepeer-review

18 Citations (Scopus)

Abstract

A bacterial expression system for the variable region fragments (Fvs) of the anti-MUC1 tumor antigen antibody MUSE11 has been constructed. The Fv fragment showed binding specificity toward TFK-1 cells, with slightly reduced affinity compared to its parent IgG. The single-chain Fv fragment was arranged in two orders, VH-linker-VL and VL-linker-VH. However, linking the regions with a flexible peptide linker (GGGGS)3 or with a shorter linker (GGGGS) led to a dramatic decrease in the biological activity toward the target antigen in both arrangements, suggesting that the MUSE11 antibody loses its activity when the domains are linked with polypeptide linkers. These results indicate that the variable region domains of the anti-MUC1 antibody MUSE11 have specificity only in the Fv form, and that linking the domains strongly reduces the association with its target antigen. Gel filtration analysis indicates that the scFv has a dimeric structure, suggesting that the inactivation of MUSE11 scFv is due to unfavorable intermolecular associations of the scFv chains. To our knowledge, this is the fist report of a significant reduction in affinity caused by linking the variable domains in both arrangements, i.e., VH-VL and VL-VH.

Original languageEnglish
Pages (from-to)673-679
Number of pages7
JournalJournal of biochemistry
Volume127
Issue number4
DOIs
Publication statusPublished - 2000 Jan 1

Keywords

  • Flow cytometry
  • Fv
  • MUC1
  • Refolding
  • scFv

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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