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Protein-Binding Ligand Screening by Bacterial Display

The bacterial display is a well-established methodology for the discovery and optimization of peptides with binding functions. Although the identification of protein-binding ligands is commonly used by phage display, bacterial display is particularly well suited for yielding high-affinity protein-binding ligands. Creative Biolabs provides the unique Hi-affinity™ bacterial display technology for the screening of protein-binding ligands.

Bacterial display (also refers to bacteria display or bacterial surface display) is a protein engineering technique which displays libraries containing billions of diverse molecules polypeptides on the surface of bacteria and, subsequently, to identify rarely desired polypeptides using selection or high-throughput screening methods. Peptide libraries can be constructed in Escherichia coli as insertions in extracellular proteins (such as pili or flagella subunits) or as insertions into outer membrane proteins for screening. The bacterial display system has been approved to be simple and efficient and has been applied extensively to find the affinity of a ligand for its target protein.

In particular, bacterial display libraries have been used to screen protein-binding ligands. Creative Biolabs has developed a robust bacterial display system to allow the isolation of high-affinity binding peptides specific to arbitrary targets. We developed several bacterial display vectors which allow high-level expression of peptides without compromising cell growth rate and viability. By coupling with magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) techniques, peptides with high binding affinity for a variety of protein targets could be isolated in less than one week.

Our protein-binding ligand screening process allows real-time analysis of displayed peptide properties, including target-binding affinity, specificity, and peptide stability to proteases during screening. The ability to measure these properties enables efficient identification of peptides and allows direct evaluation of library population fitness.

We provide a wide variety of different scaffolds, or carrier proteins, to present peptides and proteins on the outer surface of E. coli, e.g. OmpA, OmpX, and CPX. The bacterial display scaffolds allow N-terminal, C-terminal, and insertional fusions.

Representative bacterial display scaffolds and their topologies Figure 1. Representative bacterial display scaffolds and their topologies. (a) Insertion scaffolds (e.g. OmpX), (b) N-terminal display scaffolds (e.g. AIDA-I autotransporter), (c) C-terminal display scaffolds (e.g. LppOmpA), and (d) combination of N-terminal and C-terminal display using circularly permuted OmpX (CPX). Arrows indicate permissive insertion or fusion sites for display. (Daugherty et al. 2007)

Although the most frequently used host for bacterial display is Gram-negative bacterium Escherichia coli, it has an additional periplasmic space, which makes it a harder task of translocating proteins. Gram-positive bacteria lack the periplasmic space, so they are more suitable for displaying peptides. Creative Biolabs investigated Gram-positive bacterial display system using Staphylococcus carnosus. Although this system has lower transformation efficiency compared to the E. coli system, such hosts may be particularly useful in screens involving harsh selection pressure (e.g. pH, proteases) to increase stringency as they have thick cell walls.

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

References

  1. Patrick S Daugherty (2007). Protein engineering with bacterial display. Current Opinion in Structural Biology 17:474–480.
  2. Bessette P H, Rice J J, and Daugherty P S (2004). “Rapid isolation of high-affinity protein binding peptides using bacterial display.” Protein Eng Des Sel 17(10):731-739.



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