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Cell-Binding Peptides Identification by Bacterial Display

Bacterial display platform offers the prospect of simple means to generate cell-specific binding peptides. The use of bacterial display provides a convenient means to identify ligands that mediate entry or invasion of target cells which can be further developed as drug targets. Creative Biolabs has established several different bacterial display systems (Invasin, FhuA, OmpA, FliTrx, and CPX) for the identification of peptide ligands that bind to different cell types.

Bacterial display (or bacteria display or bacterial surface display) is an in vitro display technique, which displays protein or peptide libraries on the surface of bacteria. Coupled with selection or high-throughput screening methods, rarely desired polypeptides can be identified. The bacterial display is often coupled with magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) techniques to analyze target-binding affinity, specificity, and peptide stability.

Cell-Binding Peptides Identification by Bacterial Display

In particular, bacterial display has been used to discover peptide ligands that bind specifically to a given cell type. One synthetically prepared peptide retains binding activity in a cell culture assay and is stable in serum for 24 hours.

Cell-binding peptides identification by bacterial display relies on FACS to screen for target cell binding by co-expression of GFP to bacterial display libraries. The binding clones are recovered from the target cells by regrowth. By “panning” of bacterial libraries on whole cells for several rounds, this system enables the identification of unique clones against many different cell types simply and efficiency.

Screening for cell-binding peptides. (A) Scatter plot (side vs. forward scatter with both on a logarithmic scale) with the detector voltages adjusted so that the bacteria and target cells are two complete and distinct populations. (B) Bacteria expressing GFP, but without a displayed peptide, show negligible binding to the target cells. (C) Bacteria expressing a receptor-specific peptide binds efficiently to the tumor cells as shown by the large increase in green fluorescence. Figure 1. Screening for cell-binding peptides. (A) Scatter plot (side vs. forward scatter with both on a logarithmic scale) with the detector voltages adjusted so that the bacteria and target cells are two complete and distinct populations. (B) Bacteria expressing GFP, but without a displayed peptide, show negligible binding to the target cells. (C) Bacteria expressing a receptor-specific peptide binds efficiently to the tumor cells as shown by the large increase in green fluorescence. (Getz et al. 2012)

To identify cell-binding peptides more accurately, Creative Biolabs has developed a control system to measure background fluorescence levels. This system including a cell sample containing only the target cells to properly set the detector voltages, a sample containing only bacteria to locate of the bacteria on the FSC-SSC plot, and a sample of cells with GFP-expressing bacteria that express only the display scaffold (no displayed peptide) to determine the extent to which the bacterial surface interacts with the cells. With an efficient cell-binding peptide, it is possible for 10 or more bacteria to bind to an individual cell.

Quantitative Bacterial Display Library Screening

Creative Biolabs can enable quantitative screening via FACS for cell-specific ligands. We have developed a display system that encoding a random peptide library, which displays with the second cistron encoding a fluorescent protein. Thus, peptide-displaying bacterial cells binding to the target cells are quantified by using FACS.

For more detailed information, please feel free to contact us or directly sent us an inquiry.

References

  1. Getz JA, Schoep TD and Daugherty PS (2012). “Peptide discovery using bacterial display and flow cytometry.” Methods Enzymol 503:75-97. doi: 10.1016/B978-0-12-396962-0.00004-5.
  2. Patrick S Daugherty (2007). Protein engineering with bacterial display. Current Opinion in Structural Biology 17:474–480.



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