• Purity

  >90%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to cleave beta -casein. Recombinant     E. coli HTRA/Protease Do will cleave >95% cleavage of beta -casein, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  E. coli-derived Ala27-Gln474, with a C-terminal 10 His tag and ERSIGS sequence

• Accession #

• N-terminal Sequence    
  Analysis

  Ala27

• Predicted Molecular Mass

  49 kDa

• SDS-PAGE

  50 kDa, 41 kDa and 27 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM Tris, pH 8.0

• Recombinant E.coli HTRA/Protease Do (reHTRA) (Catalog # 1670-SE)

• beta -Casein (Sigma, Catalog # C-6905), 1.0 mg/mL stock in 25 mM Tris, 0.15 M NaCl, pH 7.5

• SDS-PAGE with silver staining

1. Dilute beta -Casein to 0.4 mg/mL in Assay Buffer.

2. Dilute reHTRA to 0.04 mg/mL in Assay Buffer.

3. Combine 25 μL of beta -Casein and 25 μL of reHTRA in a tube. Prepare a control containing 25 μL of beta -Casein and 25 μL of Assay Buffer.

4. Incubate the mixture for 1 hour at 45 °C.

5. Stop the reaction by adding SDS-PAGE sample buffer.

6. Analyze the cleavage by SDS-PAGE using silver staining to detect protein.

Per Reaction:

• reHTRA: 0.02 mg/mL

• beta -Casein: 0.2 mg/mL

Background: HTRA/Protease Do

As a heat shock protein and a serine protease, HTRA (Protease Do or the DegP protease) is indispensable for bacterial survival at temperatures above 42 °C (1). It is a periplasmic protein with a signal peptide of 26 amino acid residues. The mature protein contains two 90 amino acid PDZ domains, which start at residue 280. The serine protease activity is inhibited by diisopropyl fluorophosphate or dichloroisocoumarin. Four human proteins have been identified that are homologous to E. coli HTRA and believed to play a role in arthritis, cell growth, unfolded stress response, apoptosis, aging, and tumor progression (2).

• References:

1. Skorko-Glonek, J. et al. (1995) J. Biol. Chem. 270:11140.

2. Clausen, T. et al. (2002) Mol. Cell 10:443.

• Long Name:

  High Temperature Requirement

• Entrez Gene IDs:

  962111 (E. coli)

• Alternate Names:

  DegP; HTRA; Protease Do