Phage Display Protease Profiling via Flanking Affinity Tags
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Solution Overview
Problem
Current methods for unbiased measurement of protease activity are limited by high costs, low throughput, and complexity, particularly in characterizing protease activities in complex biological samples, leading to gaps in knowledge about active proteases in diseases and their substrates.
Innovation Solution
The development of SEPARATE, a bacteriophage display system using a human proteome library with monovalent phage display of 90-aa peptides, allowing for efficient and unbiased profiling of protease activities through immobilization and recapture of phage particles using flanking tags, enabling detection of both well-characterized and novel substrates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If mass spectrometry based profiling is used to measure protease activity, then unbiased measurement is achieved, but cost increases and throughput decreases
Solution Approach 1:
The patent uses peptide libraries displayed on phage particles as simplified copies of actual protein substrates. These peptide copies capture the essential cleavage sites and can be synthesized and displayed in large quantities, enabling high-throughput screening while maintaining the ability to identify genuine protease substrates through sequence matching to the proteome
Solution Approach 2:
The patent replaces the complex mass spectrometry detection system with a simpler phage display-based detection system. Instead of using expensive and complex MS instrumentation to detect proteolytic products, the system uses phage particle display and sequencing to identify cleavage events, dramatically reducing cost and increasing throughput while maintaining measurement capability
2Measurement precision
If multiplexed peptide substrate based measurements are used, then protease activity can be measured, but cost increases and throughput remains low
Solution Approach 1:
The patent creates a universal phage display platform that can screen for multiple protease activities simultaneously using a single comprehensive peptide library. The library is designed to cover the entire human proteome, allowing one assay to identify substrates for multiple different proteases at once, achieving true multiplexing without the cost and complexity of individual substrate assays for each protease
Solution Approach 2:
The patent changes the fundamental parameters of the assay system by moving from synthesized peptide substrates to phage-displayed peptide libraries. This parameter change enables combinatorial screening of thousands of peptides in parallel, transforming the throughput from low (individual peptide assays) to high (library-scale screening), while the sequencing-based detection maintains measurement precision
3Duration of action of moving object
If cell based assays are used to monitor real-time proteolytic activities, then real-time monitoring is achieved, but multiplexing capability is limited to 2-3 substrates
Solution Approach 1:
The patent uses phage particles displaying peptide sequences as simplified copies of cellular protein substrates. These peptide copies can be screened in large numbers without the complexity of maintaining living cells, yet still provide information about real proteolytic activities by matching identified cleavage sites to known protein sequences in the proteome
Solution Approach 2:
The patent replaces the complex cellular system with a simpler in vitro phage display system. Instead of using living cells that can only accommodate 2-3 substrates due to space and complexity constraints, the system uses phage particles that can display thousands of different peptide sequences simultaneously, dramatically increasing multiplexing capability while maintaining the ability to monitor proteolytic activities
4Measurement precision
If phage display of random peptide libraries is used to characterize recognition motifs, then sensitivity is achieved, but the approach cannot be generalized to unbiased characterization of protease activities in complex samples
Solution Approach 1:
The patent changes the library composition parameter from completely random peptides to a proteome-informed library design. The library is constructed to represent the actual human proteome sequence space, ensuring that displayed peptides are derived from real protein sequences. This parameter change maintains the sensitivity of random library screening while adding the versatility to identify genuine physiological substrates by matching cleavage sites to the known proteome
Solution Approach 2:
The patent creates a universal screening platform using a comprehensive human proteome peptide library displayed on phage. This single library can be used to screen for substrates of any human protease, making the approach universally applicable to all proteases rather than requiring separate assays for each enzyme. The library's comprehensive coverage of human protein sequences enables unbiased characterization of protease activities in complex samples
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
SEPARATE facilitates low-cost, high-throughput characterization of protease activities, identifying novel substrates like HUWE1 and detecting known substrates at physiological concentrations, thus overcoming limitations of existing methods and providing insights into proteolytic processes in health and disease.
Implementation Method 1
immobilization and recapture of phage particles using flanking tags
Implementation Method 2
profiling protease activity using phage display
Data Source
AI summary
The present invention relates to the field of proteases. More specifically, the present invention provides compositions and methods useful for profiling protease activity using phage display. In one embodiment, a display vector useful for profiling protease activity comprises a nucleic acid sequence encoding (a) a peptide to be displayed on the surface of the vector; (b) a first affinity tag C-terminal to the peptide; and (c) a second affinity tag N-terminal to the peptide. The display vector can comprise a virus, bacteriophage, yeast, bacteria, retrovirus, ribosome or mRNA. In particular embodiments, the peptide comprises a human peptidome library peptide.