Creative BioMart is a well-known expert who committed to developing a variety of methods to explore enzyme sequences and create new high-efficiency biocatalysts. With years of experience, we provide customized protease substrate specific analysis services and identify the best cleavage site of the protease to precisely meet customer requirements.

Introduction of Protease Substrate Specificity

Proteolysis is one of the most common post-translational modifications (PTM) and plays an important role in all aspects of cell function. By adding phosphoryl groups to the serine, threonine or tyrosine residues of the substrate protein, kinases can regulate processes such as signal, cell cycle, and gene expression. About 600 human proteases (approximately 2% of the genome) work together to maintain the normal function and homeostasis of cells and tissues in the body. Abnormal protease activity can spread cancer, inflammation and infectious diseases. Many kinase mapping technologies currently use fixed peptide arrays or multi-well plate peptide pools, allowing the generation of specific motifs. In order to potentially screen a larger and more diverse sequence space, researchers have developed genetically encoded substrate phage or yeast display libraries. Substrate profiling is a process of rapid detection of purified proteins or complex biological mixtures with different substrates to study the substrate specificity of these biocatalysts. Protease substrate specificity is a powerful tool for biochemical proteomics, especially because many sequence and structurally related proteins can perform distinct chemical reactions.

Fig 1. A Chemical Strategy for Protease Substrate Profiling. (Griswold AR, et al., 2019)

Services

Understanding the substrate specificity of proteases helps to define the function of proteases in cellular processes and provides insights for the design of inhibitors for research and therapeutic purposes. As a leading protein engineering service provider, Creative BioMart is dedicated to protease substrate specific analysis services, aiming to identify the best cleavage site of protease and extensive identification of natural substrates.

The wide identification of optimal cleavage sites and natural substrates for proteases is essential for drug discovery and understanding of protease biology. Proteomics methods allow people to understand more extensively the protease substrates on intact proteins, but they require large amounts of lysates and miss low-abundance proteins and those that are not expressed at all in the cell lines tested. Our scientists have developed a rapid, reproducible, easy to perform and analyze, low-cost and reproducible, with unprecedented substrate sequence coverage depth for protease specific analysis. We combined the substrate phage display library with next-generation sequencing (SPD-NGS), which can analyze the protease specificity of 10³ to 10⁴－It is twice as deep as the classical synthetic peptide library and identified specific human sequences that can be cleaved. With our experience in enzyme engineering, we provide a professional one-stop service:

• Substrate detection design and verification
• Substrate phage library construction
• Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) determination
• Sequence ID recognized by SPD-NGS
• Predict new protein substrates
• Data analysis and development planning

Creative BioMart conducts a more in-depth analysis of the specificity of protease substrates to identify consensus linear sequences and even candidate protein substrates in an unprecedented range of proteome. We will work with you to develop the most appropriate strategy and provide the most meaningful data for your research for accelerating the research of life sciences. If you are interested in our services, please do not hesitate to contact us for more information.

References

1. Zhou J, Li S, Leung KK, et al.. (2020) Deep profiling of protease substrate specificity enabled by dual random and scanned human proteome substrate phage libraries. Proc Natl Acad Sci U S A. 117(41): 25464-25475.
2. Griswold AR, Cifani P, et al.. (2019) A Chemical Strategy for Protease Substrate Profiling. Cell Chem Biol. 26(6): 901-907.e6.

• Enzyme Activity Analysis Service
• Enzyme Stability Enhancement Service
• Protease Substrate Screening Service