A substrate phage enzyme-linked immunosorbent assay to profile panels of proteases

It is estimated that proteases comprise nearly 2% of the human genome. Give n that the primary structure of all known proteases will soon be available, an important challenge is to define the structure-activity relationships t hat govern substrate hydrolysis. Ideally this would be accomplished on a ge nome-wide scale. To this end, we have developed a one-pot phage selection s ystem that yields the substrate recognition profile of multiple proteases f rom a single round of selection. The system meets five key criteria: (i) mu ltiple proteases can be analyzed simultaneously, (ii) prior knowledge of su bstrate preference is not required, (iii) information regarding substrate p references on both side of the scissile bond is obtained, (iv) the system y ields selective substrates that distinguish closely related proteases, and (v) semiquantitative information on substrate hydrolysis is obtained, allow ing for the assignment of initial rank-order preferences. As an illustratio n, a phage selection with a mixture of thrombin and factor Xa (serine prote ases) along with matrix metalloproteinase and atrolysin C (metalloproteinas es) was performed. Peptide substrates were identified that (i) have high k( cat)/K-m ratios, (ii) are selective for individual proteases, and (iii) mat ch the sequences of known physiological substrates. (C) 2001 Academic Press .