DegP periplasmic protease a new anti-infective target and an in vitro assay for DegP protease function

US6306619A · kind A · utility

Assignees

• Washington University · US
• SIGA Pharmaceuticals, Inc. · US

Inventors

• Hal C. Jones · Corvallis, US
• Christopher Matthew Liu · Cambridge, US
• Scott Hultgren · Ballwin, US
• Dennis E. Hruby · Albany, US
• Christine A. Franke · Albany, US
• Amy Evans · West Linn, US

Key dates

• Filing date: Jun 29, 2000
• Grant date: Oct 23, 2001
• Expiry date: Jun 29, 2020

Classification

• Technology area (CPC G): Physics
• CPC primary: G01N33/573
• WIPO field: Measurement
• WIPO sector: Instruments

Abstract

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 NH.sub.2 -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…