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, 96 (11), 6025-30

Making Artificial Antibodies: A Format for Phage Display of Combinatorial Heterodimeric Arrays

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Making Artificial Antibodies: A Format for Phage Display of Combinatorial Heterodimeric Arrays

C Gao et al. Proc Natl Acad Sci U S A.

Abstract

The gene VII protein (pVII) and gene IX protein (pIX) are associated closely on the surface of filamentous bacteriophage that is opposite of the end harboring the widely exploited pIII protein. We developed a phagemid format wherein antibody heavy- and light-chain variable regions were fused to the amino termini of pVII and pIX, respectively. Significantly, the fusion proteins interacted to form a functional Fv-binding domain on the phage surface. Our approach will be applicable to the display of generic peptide and protein libraries that can form combinatorial heterodimeric arrays. Consequently, it represents a first step toward artificial antibodies and the selection of novel biological activities.

Figures

Figure 1
Figure 1
Filamentous phage fd architecture.
Figure 2
Figure 2
Gene construct of the phagemid pCGMT-1b used for Fv display.
Figure 3
Figure 3
Phage ELISA of the different Flag and pVII or pIX fusion proteins.
Figure 4
Figure 4
Phage ELISA of the antigen (PCP-BSA)-binding capacities of the different fusion constructs of the 21H3 VH and VL with pVII and pIX.
Figure 5
Figure 5
Electron micrographs showing antigen-specific labeling of filamentous phage displaying the 2H6 Fv heterodimer. Similar results were obtained for the 21H3 phage Fv; however, the micrographs were not as distinct. (A) A phage specifically labeled with a 5-nm colloidal gold particle adhered to the PCP-BSA antigen (×105,000). (B) A phage on the same grid as in A labeled with two gold particles (×105,000).
Figure 6
Figure 6
Enrichment of anti-PCP 2H6 phage Fv during panning. Pooled phage eluted after each round were amplified, and 1010 cfu were added to each well of a microtiter plate precoated with PCP-BSA or GNC-BSA. Phage were detected by using an anti-M13 antibody as described (Materials and Methods).

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