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Protease Substrate Identification by Bacterial Display

The use of bacterial display provides a convenient means to identify peptide substrates for proteolytic enzymes. Creative Biolabs has established CLiPS system for the identification of optimal peptide substrates of protease.

Bacterial display, also called bacteria display or bacterial surface display, is an advanced protein engineering technique. By this technique, libraries containing billions of diverse molecules polypeptides can be displayed on the surface of bacteria to screening target peptides or proteins. Bacterial display is often coupled with magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) techniques which allow real-time analysis of many properties (e.g. target-binding affinity, specificity, stability) of proteins during screening.

Proteolytic Enzymes

Proteolytic enzymes have remarkable selectivity for protein and peptide substrates. Substrate selectivity can be achieved through a variety of mechanisms, including the use of distinct exosites, secondary substrate binding domains or by recognizing extended linear substrates at the active site. Phage display is the most commonly used method for protease substrate identification. Other substrate specificity characterization methods including positional scanning, oriented peptide libraries, and cellular libraries of peptide substrates (CLiPS).

CLiPS for Protease Substrates Identification

A two-color bacterial display system termed cellular libraries of peptide substrates (CLiPS) has been used for protease substrates identification. eCPX scaffold bacterial display system is used in CLiPS. eCPX scaffold enables presentation of peptides on both scaffold terminus (both C-terminus and N-terminus). In this approach, a plasmid codes a streptavidin-binding peptide is fused to the N-terminus of eCPX which is upstream of the substrate library. On the C-terminus of eCPX, an affinity tag (usually SH3 domain-binding peptide) is fused to improve the resolution of scaffold expression and binding affinity, verify expression of the full-length construct, and enable two color screening for cleaved substrates. The bacteria can be incubated with both SAPE (red probe) and AlajGFP (green probe) which can be fused to the SH3 domain of monocytic adaptor protein (Mona) to separately label both termini of the eCPX construct. Screening of proteolytic substrates is performed by FACS. This method allows screening by cleavage kinetics and direct ranking of identified substrates on the cell surface. Proteolytic substrates were identified and sorted using their absence of fluorescence after incubation with enteropeptidase. The selection rounds consist of sorting for bacteria that are efficiently labeled with both the red and green probes, followed by sorting the red fluorescence decreases while the green fluorescence remains high isolates which indicates substrate hydrolysis.

Protease substrate screening using two-color CLiPS. A candidate protease substrate peptide and a red fluorescent probe peptide binder are fused to the N-terminus of CPX on the surface of the E. coli. To avoid collection of false positives, a green fluorescent probe peptide binder is fused on the C-terminus of CPX and cellular fluorescence is measured using FACS. The hydrolysis of the substrates causes the loss of the red fluorescent probe ligand, such that clones with cleaved substrates exhibit reduced red, but high green fluorescence Figure 1. Protease substrate screening using two-color CLiPS. A candidate protease substrate peptide and a red fluorescent probe peptide binder are fused to the N-terminus of CPX on the surface of the E. coli. To avoid collection of false positives, a green fluorescent probe peptide binder is fused on the C-terminus of CPX and cellular fluorescence is measured using FACS. The hydrolysis of the substrates causes the loss of the red fluorescent probe ligand, such that clones with cleaved substrates exhibit reduced red, but high green fluorescence. (Boulware et al. 2010)

Two-color CLiPS screening method Figure 2. Two-color CLiPS screening method (Boulware et al. 2010).

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References

  1. Boulware K T, Jabaiah A, and Daugherty P S (2010) “Evolutionary optimization of peptide substrates for proteases that exhibit rapid hydrolysis kinetics.” Biotechnol Bioeng 106(3):339-46. doi: 10.1002/bit.22693.
  2. Patrick S Daugherty (2007). Protein engineering with bacterial display. Current Opinion in Structural Biology 17:474–480.
  3. 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.

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