Signal sequence as a determinant in expressing disulfide-stabilized single chain antibody variable fragments (sc-dsFv) against human VEGF

Ing Chien Chen, Chung Ming Yu, Yu Ching Lee, Yi Jen Huang, Hung Ju Hsu, An Suei Yang

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Phage-displayed single chain variable fragment (scFv) libraries have been powerful tools in antibody engineering. But the scFv structures are frequently unstable due to the dissociation of the dimeric interface between the two variable domains. One solution is the sc-dsFv construct, where the single chain variable domain fragment is stabilized with an additional interface disulfide bond, leading to stable and homogeneous dimeric interface for the sc-dsFv structure. However, the phagemid system that is capable of effective expression for both sc-dsFv-pIII fusion proteins on phage surface and secreted non-fusion sc-dsFv in bacterial culture medium has not been demonstrated. In this work, a biological combinatorial approach was applied to optimize the signal sequence N-terminal to the sc-dsFv-pIII fusion protein encoded in a phagemid. The optimized sc-dsFv phage display systems were compatible with both the phage-based directed evolution procedure and the high throughput screening of the soluble sc-dsFv. The utility of the phagemid systems was demonstrated in generating anti-VEGF sc-dsFv with VEGF-binding affinity one order of magnitude higher than the corresponding scFv, due only to the interface disulfide bond in the sc-dsFv. Moreover, the protein stability of the sc-dsFv construct was unmatched by the corresponding scFv. These advantages of the sc-dsFv were gained through the interface disulfide bond of the sc-dsFv and the novel signal sequence in the phagemid.

Original languageEnglish
Pages (from-to)1307-1315
Number of pages9
JournalMolecular BioSystems
Volume6
Issue number7
DOIs
Publication statusPublished - Jun 21 2010
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Biology

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