Background

Phage display peptide libraries that display random 5-20 mer peptides have been widely used to determine protease substrate (sequence) specificity for a large number of enzymes and semi-purified cell extracts. This technique has been playing a critical role in designing inhibitors and activators of proteases.

In comparison with phage display peptide libraries used in other fields, phage display peptide libraries used to identify the preferred substrate sequences of a protease usually express a fusion tag at the N-terminus of the peptide sequences, while, like in regular phage display peptide libraries, the minor coat viral protein pIII is located at the C-terminus of the peptide sequences. The N-terminal fusion tag is intended to bind the phage particles to a solid support. Scientists from Creative Biolabs have designed and built up a unique tag, HiAffi-tag, which is derived from Protein G, thus has strong binding affinity to most IgG coated on a solid surface. In contrast to His Tag and Strepavidin Binding Motifs used in such libraries, HiAffi-tag enables the best capture of the phage particles to the solid support and the least steric hindrance for proteolysis of the substrate peptides.

During library screening against a particular protease, phage particles that express sensitive peptide sequences are cleaved off the solid support. After that, the free phages are amplified, immobilized and subjected to protease cleavage again. After several rounds of screening, selected phage clones are DNA sequenced and common features of the peptide sequences are determined. These peptide sequences are good candidates for further identification of the substrates of the protease.

Protease Substrate based Library Screening Service

Creative Biolabs also developed proprietary in silicon screening and computer aided rational design methods to use such peptide sequences, which must fit into the active site of the protease, as leads to develop inhibitors and activators of a protease.

Other optional phage display library screening services:

• In Vivo Phage Library Screening Services
• Ex vivo Phage Display Services
• Magic™ Therapeutic Antibody Discovery Service
• Solid-phase Library Screening
• Solution-sorting Library Screening
• Cell-based Library Screening
• Premade Phage Display Antibody Library Screening Service
• Premade Phage Display Peptide Library Screening Service
• Rapid Recovery of scFv

Published Data

• Discovery of Peptide Ligands Targeting a Specific Ubiquitin-like Domain-binding Site

Fig. 1 Effects of a UBL-binding site mutant on peptide recognition and homologous recombination.^{1, 2}

The research focuses on identifying peptide ligands targeting a specific binding site in the deubiquitinase USP11, a key regulator in DNA repair processes such as homologous recombination. The significance of this study lies in the discovery of unique USP11-interacting peptide motifs using a next-generation phage display (NGPD) strategy, which offers potential for developing targeted therapeutic agents and biochemical tools. The results revealed two consensus peptide motifs with high specificity for USP11, which do not interact with its paralogs USP4 and USP15. The application of protease substrate-based phage display library screening in this study was crucial for uncovering novel binding sites and ligands. This unbiased approach enabled the identification of selective peptide ligands, which could be further developed as molecular probes or therapeutic agents targeting USP11-related pathways, especially in cancer treatments that focus on DNA damage repair mechanisms.

FAQ

1. What is protease substrate-based phage display library screening

   Protease substrate-based phage display library screening is a method used to identify peptides that interact with specific proteases. Phage display involves inserting peptide sequences into bacteriophage coats, which are then screened for their ability to bind or be cleaved by a target protease. This method helps identify substrates or inhibitors, providing insight into protease function and potential therapeutic targets.

2. How does protease substrate-based phage display work

   In protease substrate-based phage display, libraries of random peptides are displayed on the surface of bacteriophages. These phages are exposed to a specific protease, and peptides that bind to or are cleaved by the protease are selected. This approach enables high-throughput identification of protease substrates, which can aid in understanding protease activity and developing protease inhibitors.

3. What are the advantages of using phage display for protease substrate screening

   Phage display offers several advantages for protease substrate screening, including its high-throughput nature, ability to screen millions of peptide variants, and potential to uncover novel interactions. The technique allows the identification of both substrates and inhibitors, making it a versatile tool for drug discovery, particularly for targeting proteases involved in diseases such as cancer and inflammation.

4. What applications are there for protease substrate-based phage display screening

   This screening method has wide-ranging applications, including drug discovery, understanding protease functions in diseases, and developing diagnostic tools. In cancer research, it helps identify protease substrates involved in tumor progression. In infectious diseases, it can uncover protease targets in pathogens, aiding in the design of specific inhibitors or therapeutic agents.

5. How is data analyzed in protease substrate-based phage display screening

   Data from protease substrate-based phage display screening is analyzed using bioinformatics tools that process the peptide sequences identified during screening. Sequencing of selected phages helps determine which peptide sequences interact with the protease. Statistical analysis, such as motif enrichment, is used to pinpoint consensus sequences that act as substrates or inhibitors, guiding further validation and research.

6. Can protease substrate-based phage display screening be used in therapeutic development

   This screening method is highly valuable in therapeutic development, particularly for designing protease inhibitors. By identifying substrates that interact with proteases involved in diseases such as cancer or neurodegenerative disorders, researchers can develop inhibitors that block these interactions, leading to potential drug candidates. This technique accelerates the discovery of novel, targeted therapies.

7. How does protease substrate-based phage display contribute to personalized medicine

   Protease substrate-based phage display can contribute to personalized medicine by identifying protease substrates specific to an individual's disease profile. For example, in cancer, identifying tumor-specific proteases and their substrates allows for the development of tailored inhibitors or diagnostic markers. This approach can lead to more effective, targeted treatments based on the patient's unique protease activity profile.

Resources

Use the resources in our library to help you understand your options and make critical decisions for your study.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.