DeltaPhage--a novel helper phage for high-valence pIX phagemid display

Abstract

Phage display has been instrumental in discovery of novel binding peptides and folded domains for the past two decades. We recently reported a novel pIX phagemid display system that is characterized by a strong preference for phagemid packaging combined with low display levels, two key features that support highly efficient affinity selection. However, high diversity in selected repertoires are intimately coupled to high display levels during initial selection rounds. To incorporate this additional feature into the pIX display system, we have developed a novel helper phage termed DeltaPhage that allows for high-valence display on pIX. This was obtained by inserting two amber mutations close to the pIX start codon, but after the pVII translational stop, conditionally inactivating the helper phage encoded pIX. Until now, the general notion has been that display on pIX is dependent on wild-type complementation, making high-valence display unachievable. However, we found that DeltaPhage does facilitate high-valence pIX display when used with a non-suppressor host. Here, we report a side-by-side comparison with pIII display, and we find that this novel helper phage complements existing pIX phagemid display systems to allow both low and high-valence display, making pIX display a complete and efficient alternative to existing pIII phagemid display systems.

Figure 1.

(A) Schematic illustration of the pV, pVII, pIX and pVIII genomic region of M13 important for the DeltaPhage design. The modification (bold, italic, underlined) was inserted between residue two and three in the pIX ORF downstream of the pVII translational stop overlapping with the pIX translational start. The region is framed by the unique BsrGI/SnaBI restriction sites shared by the fd and M13 genomes. (B) Basic outline of phagemid rescue using the standard helper phage M13K07, which renders low-valence display independent of supE genotype of the producing host. The phagemid is selectively propagated as a plasmid in E. coli, as it carries an ampicillin resistance marker. For phage assembly to occur, the host is super-infected with a helper phage containing all elements necessary for new virion assembly. As the helper phage also produces the wt pIX found as a POI-pIX fusion protein on the phagemid, the resulting phages carries a blend of both rendering low-valence display. (C) Basic outline of phagemid rescue using the DeltaPhage helper phage, which renders high-valence pIX display in a supE producing host due to complete lack of wt pIX production.

Figure 2.

Host-cell propagation and phage assembly characteristics of DeltaPhage. (A) Single colonies of either E. coli XL1-Blue or TOP10F′ harboring either DeltaPhage, or M13K07 as control, were grown ON in selective medium before the cell density was measured at A_600nm (values shown are extrapolated from measurements in one-fourth dilutions). The results are given as mean ± SD of three independent colonies of each sample assessed in parallel. (B) The M13K07 and DeltaPhage phage content in the supernatants from small-scale ON cultures were determined by infectious titration and the results given as the average kanamycin resistant colony forming units/ml (cfu^kanR/ml). The results are given as mean ± SD of three independent colonies of each sample assessed in parallel. ND = not detected. (C) The VCSM13, DeltaPhage and M13K07 phage content in PEG concentrated samples derived from up-scaled parallel cultures determined by either infectious titer, or total virion content determined by OD using the formula (((A_269nm – A_320nm) × 6.083 × 10¹⁶)/genome size = virions/ml) (38). Genome sizes: VCSM13, 8669 bp; M13K07, 8669 bp; DeltaPhage, 8678 bp. The results are given as mean ± SD of four to six independent experiments. (D) Agarose gel analysis of intact helper phage virions as described in ‘Materials and Methods’ section. The picture is representative of three experiments employing three independently packaged phage samples of both M13K07 (K07) and DeltaPhage (Delta).

Figure 3.

Assessment of DeltaPhage in pIX display phagemid rescue. (A) Two pIX display phagemids (encoding either an anti-phOx or anti-NIP scFv) were propagated in 50 ml cultures of E. coli XL1-Blue and TOP10F′ followed by rescue by either M13K07 or DeltaPhage helper phages. Phages were precipitated and concentrated by PEG, followed by infectious titration and the results given as cfu^ampR/ml. (B) Phagemid to helper phage ratios were determined by cfu^ampR/cfu^kanR from parallel infectious titration of the same samples. The results are given as mean ± SD of three independent colonies of each sample assessed in parallel. (C) Two pIII display phagemids (encoding either the anti-phOx or anti-NIP scFv) were propagated in E. coli XL1-Blue and rescued with either M13K07, or Hyperphage. Phagemid to helper phage ratios were determined as above. The results are given as mean ± SD of three to four independent colonies of each sample assessed in parallel.

Figure 4.

Functional analysis of pIX display levels at low and high-valence display. Serial dilutions of phagemid-rescued samples displaying either scFv anti-phOx (A), scFv anti-NIP (B) or (C) Fab anti-phOx were applied in a phage capture ELISA as described in ‘Material and Methods’ section. Antigen-bound phages were detected by anti-M13^HRP and the data shown as function of number of phagemids (cfu^ampR)/ml solution. The data is representative of three to four independent experiments.

Figure 5.

Phage staining of 4B2A1 T cells by flow cytometry. A normalized phage input of 5 × 10¹¹ cfu/ml, which corresponds to a concentration of 830 pM was used throughout. A total of 2 × 10⁵ T cells were incubated with the indicated phages followed by detection using anti-fd^bio and SA–PE as described in ‘Materials and Methods’ section. Cells were also stained with the 4B2A1 clonotype-specific mAb GB113. The samples were analyzed on a FACScalibur flow cytometer and the histograms showing staining on the cognate 4B2A1 T cells (A), or the negative control T cells 7A10B2 (B) were generated using CellQuest Pro (BD Biosciences). (C) SDS–PAGE/western blot analysis of the phage samples tested in flow. Normalized phage samples of 2 × 10⁹ cfu^ampR/lane were used and detected using an anti-pIII antibody as described in ‘Materials and Methods’ section. Key to figure: K, M13K07-rescued phagemid; H, Hyperphage-rescued phagemid; D, DeltaPhage-rescued phagemid; C, M13K07.