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DegP periplasmic protease a new anti-infective target and an in vitro assay for DegP protease function
DegP periplasmic protease a new anti-infective target and an in vitro assay for DegP protease function
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机译:DegP周质蛋白酶,一种新的抗感染靶标和DegP蛋白酶功能的体外测定
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摘要
The DegP (HtrA) protease is a multifunctional protein essential for the removal of misfolded and aggregated proteins in the periplasm. The present invention provides an assay for inhibitors of DegP activity, comprising mixing a suspected inhibitor of DegP activity with DegP and a suitable substrate (preferably a native substrate of DegP such as PapA) and detecting changes in DegP activity. DegP has been shown to be essential for virulence in several Gram negative pathogens. Only three natural targets for DegP have been described: colicin A lysis protein (Cal), pilin subunits (K88, K99, Pap) and recently HMW1 and HMW2 from Hemophilus influenzae. In vitro, DegP has shown weak protease activity on casein and several other non-native substrates. The present inventors have identified the major pilin subunit of the Pap pilus, PapA, as a native DegP substrate and demonstrated binding and proteolysis of this substrate in vitro. Using an NH2-terminal affinity tag the present inventors have purified PapA away from the PapD chaperone, in the presence of denaturant, to use as a proteolysis substrate. This finding will allow the identification of the DegP recognition and cleavage sites in substrate proteins, and further, allow the design of small molecule inhibitors of protease function.
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