Phage display of disulfide-stabilized Fv fragments.
Phage display of single-chain Fvs (scFvs) is a powerful tool to enrich and isolate specific antibody fragments from large pools (libraries) of Fv coding genes. However, many scFvs and scFv fusion proteins are unstable, not only as soluble proteins but also on the surface of phage. This limits and biases the recovery of specific Fv phage from display libraries to relatively stable scFvs. Also, the peptide linker in scFvs can diminish antigen binding of scFvs and scFv-fusion proteins. Disulfide-stabilized Fvs (dsFv) which have the VH-VL heterodimer stabilized by an interchain disulfide bond connecting framework regions in VH and VL rather than a peptide linker are more stable than scFvs and in some instances show better binding. To analyze whether these advantages can be utilized in a phage display system and to prove the feasibility of dsFv display, we constructed phage for dsFv display of the anti-Tac antibody and a dsFv-phage library. We find that dsFv phage can specifically bind antigen although the titer of dsFv phage in supernatants appears to be reduced compared to scFv phage. But this reduction in titer does not hamper the isolation of dsFv phages from large pools (libraries) as demonstrated by 'panning' of anti-Tac scFv and dsFv phages on living leukemia cells in suspension. In addition, dsFv phage are more stable than scFv phage. Therefore, display of dsFvs on phage is a useful alternative and addition to scFv-phage display.
Brinkmann
U
U
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.
Chowdhury
P S
PS
Roscoe
D M
DM
Pastan
I
I
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004220:Disulfides; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D015375:Receptors, Interleukin-2 / Q000276:immunology; D014407:Tumor Cells, Cultured
D004220:Disulfides; D007135:Immunoglobulin Variable Region; D015375:Receptors, Interleukin-2