Engineering anti-vascular endothelial growth factor single chain disulfide-stabilized antibody variable fragments (sc-dsFv) with phage-displayed sc-dsFv libraries.
Phage display of antibody fragments from natural or synthetic antibody libraries with the single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in antibody engineering. However, the nature of the artificial single chain constructs results in unstable proteins expressed on the phage surface or as soluble proteins secreted in the bacterial culture medium. The stability of the variable domain structures can be enhanced with interdomain disulfide bond, but the single chain disulfide-stabilized constructs (sc-dsFv) have yet to be established as a feasible format for bacterial phage display due to diminishing expression levels on the phage surface in known phage display systems. In this work, biological combinatorial searches were used to establish that the c-region of the signal sequence is critically responsible for effective expression and functional folding of the sc-dsFv on the phage surface. The optimum signal sequences increase the expression of functional sc-dsFv by 2 orders of magnitude compared with wild-type signal sequences, enabling the construction of phage-displayed synthetic antivascular endothelial growth factor sc-dsFv libraries. Comparison of the scFv and sc-dsFv variants selected from the phage-displayed libraries for vascular endothelial growth factor binding revealed the sequence preference differences resulting from the interdomain disulfide bond. These results underlie a new phage display format for antibody fragments with all the benefits from the scFv format but without the downside due to the instability of the dimeric interface in scFv.
Huang
Yi-Jen
YJ
Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
Chen
Ing-Chien
IC
Yu
Chung-Ming
CM
Lee
Yu-Ching
YC
Hsu
Hung-Ju
HJ
Ching
Anna Tung Ching
AT
Chang
Hung-Ju
HJ
Yang
An-Suei
AS
D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D021382:Protein Sorting Signals / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D004220:Disulfides; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A