Design of helical proteins for real-time endoprotease assays

Proteases play a key role in cellular biology and have become priority targ ets for new pharmaceuticals. Thus, there is a high demand for specific, sen sitive, and quick assays to monitor the activity of endoproteases. We desig ned affinity-tagged helical proteins with unique protease cleavage sites an d thus constructed universal, molecularly defined, and uniform substrates f or in vitro detection of IgA endoprotease. The substrate is a 10.5-kDa reco mbinant helical protein with a high-affinity (His)(6)-tag at the aminotermi nal end. Further elements are a unique proteolytic recognition site and a C -terminal helical extension, which is cut off by the protease. Proteolytic action can be monitored in real time using surface plasmon resonance spectr oscopy. Femtomole amounts of protease could be reliably and quantitatively detected within a few minutes after the start of the reaction. The detectio n signal changed linearly with the amount of protease and was independent o f the applied sample flow rate. The biochip can be reversibly loaded with t he recombinant protease substrate, so that the SPR assay is well-suited for automation. By substituting an HIV protease site for the recognition site of the IgAse, we also obtained a substrate for the quantitative and sensiti ve detection of HIV-1 endoprotease. Our substrate design is thus generally applicable. (C) 2000 Academic Press.