Fig 1.
Genotypes and phenotypes of phage particles obtained with the helper cell system.
Helper cells contain one of the following helper plasmids: M13cp (full-length gene 3), M13cp-CT (truncated gene 3), or M13DG3 (fully deleted gene 3). E. coli containing random-peptide-encoding phagemids and any one of the helper plasmids produce phagemid particles expressing varying levels of peptide-fused gene 3 protein. We found that M13cp cells are highly transformation efficient, making them good candidates for generating primary libraries based on the gene 3 protein display vector pDAN5 [11] while both M13cp-CT and M13DG3 cells are highly infectable, produce very high numbers of phagemid particles, and are useful to produce amplified (secondary) libraries which have the features of monovalent or multivalent display, respectively.
Fig 2.
PCR analysis of scFv selections amplified with either helper phage or helper cells.
Extensive characterization of helper cells derived from various combinations of helper plasmids and E. coli strains led us to choose those best suited for high transformation efficiency, high infectability and high phage-producing. Toward thorough characterization of helper cells we further investigated their utility for scFv selection. The result of the PCR and capillary electrophoresis analysis of representative number of scFv clones are shown. The DNA encoding full-length scFv correspond to the higher bands (about 800 bp), whereas the lower bands correspond to DNA encoding truncated scFv of different sizes. After the second cycle of panning, library outputs amplified with either helper phage (A) or with helper cells (B) contained approximately 4% or 66% truncated scFvs respectively. Thus, helper cells are intolerant of the scFv, leading to truncations, and instead are ideal for peptide selections.
Fig 3.
Vector map and primers used for library construction.
The forward primers (FW) incorporated the different sets of random peptide sequences. The random sequences are coded by NNK, where N = A,T,C, or G and K = G or T.
Table 1.
Actual vs. theoretical percentage of each amino acid in peptide helper cell secondary libraries and commercial peptide libraries.
Fig 4.
Two peptide clones recognize live whole cell Y. pestis as well as purified F1V antigen.
Phage supernatants produced from clones randomly picked from selection outputs were assayed for binding to F1V-coated, whole cell-coated and uncoated wells. CT4 control is an scFv identified as positive from a previous selection on F1V [23]. Phage binding was detected with anti-M13-HRP conjugated antibody. The signal obtained with uncoated wells was subtracted from the signal corresponding to coated wells, and the results were normalized based on phage concentration.
Table 2.
Consensus sequences in peptides selected from linear libraries against purified F1V antigen.
Fig 5.
Binding curves for selected QBP-binding peptides.
Selected peptides were yeast-expressed and assayed by flow cytometry for binding to apo (Q) or glutamine-bound (QQ) L-glutamine-binding periplasmic protein QBP at various concentrations. Experiments were executed in duplicate and the data points in each graph are averages of the mean APC fluorescence with corresponding standard deviations. The estimated affinities from this data are D6 = 35 nanoM, F5 = 300 nanoM, and P11 = 2 microM.
Table 3.
Sequence analysis of selected QBP or streptavidin-binding peptides.