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.
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