Figure 1. Generation of filamentous M13 phages displaying cell-penetrating 3D8 VL transbody (3D8 VL-M13) and TAT peptide (TAT-M13).

As a control, anti-DR4 hAY4 scFv without cell-penetrating ability was also employed; the phage particles are designated as hAY4 scFv-M13. (A) Phage titers obtained from 100-ml culture supernatants of recombinant phagemid-transformed bacteria by VCSM13 helper phage superinfection under optimal culture conditions as described in the text. Phage titers were determined by CFU assay. Data represent mean ± S.E. (error bars) of 5 independent experiments. (B) Western blot analysis of display efficiency of fusion proteins (insert-pIII) (filled arrow) versus full-length pIII (open arrow) from VCSM13 helper phage on the recombinant M13 filamentous phage particles. Equal titers (10⁹ or 10¹⁰ CFU) of phage particles prepared as described in (A) were western blotted with anti-myc antibody to detect only pIII-fusion proteins (3D8 VL-pIII, and TAT-pIII, and hAY4 scFv-pIII) from the phagemid vectors or anti-M13 pIII antibody to detect both pIII-fusion proteins from the phagemid vectors and full-length pIII from the helper phage. The positions of molecular size marker are indicated. (C) Phage ELISA on DR4 or DNA to examine antigen-binding specificity of the recombinant phages. Various titers (10⁷∼10¹¹ CFU) of recombinant phages or VCSM13 helper phage were applied to each well, precoated with the indicated antigen. Bound phages were detected with HRP-conjugated anti-M13 antibody. Data represent mean ± S.E. (error bars) of three independent experiments carried out in triplicate.