Proteases are tightly regulated by specific inhibitors, such as serpins, which are able to undergo considerable and irreversible conformational changes in order to trap their targets. There has been a considerable effort to investigate serpin structure and functions in the past few decades; however, the specific interactions between proteases and serpins remain elusive. In this chapter, we describe detailed experimental protocols to determine and characterize the extended substrate specificity of proteases based on a substrate phage display technique. We also describe how to employ a bioinformatics system to analyze the substrate specificity data obtained from this technique and predict the potential inhibitory serpin partners of a protease (in this case, the immune protease, granzyme B) in a step-by-step manner. The method described here could also be applied to other proteases for more generalized substrate specificity analysis and substrate discovery.
|Title of host publication||Methods in Enzymology, Volume 501: Serpin Structure and Evolution|
|Editors||James C Whisstock, Philip I Bird|
|Place of Publication||USA|
|Pages||237 - 273|
|Number of pages||37|
|Publication status||Published - 2011|
Song, J., Matthews, A., Reboul, C. F., Kaiserman, D., Pike, R. N., Bird, P. I., & Whisstock, J. C. (2011). Predicting serpin/protease interactions. In J. C. Whisstock, & P. I. Bird (Eds.), Methods in Enzymology, Volume 501: Serpin Structure and Evolution (pp. 237 - 273). Academic Press. https://doi.org/10.1016/B978-0-12-385950-1.00012-2