Issue 7, 2010

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

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.

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

Supplementary files

Article information

Article type
Method
Submitted
08 Oct 2009
Accepted
04 Mar 2010
First published
27 Apr 2010

Mol. BioSyst., 2010,6, 1307-1315

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

I. Chen, C. Yu, Y. Lee, Y. Huang, H. Hsu and A. Yang, Mol. BioSyst., 2010, 6, 1307 DOI: 10.1039/B921106C

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