Recombinant Mouse Renin Protein, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse Renin Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate 5-FAM/QXL™ 520. Peti-Peterdi, J. et al. (2009) Physiology 24:88. The specific activity is >20 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Renin protein
  Leu22-Arg402, with a C-terminal 10-His tag
• Accession #: P06281
• N-terminal Sequence: Leu22
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Ren1
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 43 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 45-55 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in MES and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Activation Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl₂, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Assay Buffer: 50 mM NaH₂PO₄, 1 M NaCl, pH 7.5
  • Recombinant Mouse Renin (rmRenin) (Catalog # 4277-AS)
  • Trypsin (Sigma, Catalog # T-1426)
  • AEBSF (Catalog # EI001), 100 mM stock in DMSO
  • Substrate: “Rat Renin FRET” 5-FAM/QXL™520 (AnaSpec, Catalog # 62334), 100 µM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Activate rmRenin at 0.1 mg/mL with Trypsin at 2 µg/mL.
  1. Dilute rmRenin to 0.2 mg/mL in Activation Buffer.
  2. Dilute Trypsin to 4 µg/mL in Activation Buffer.
  3. Mix equal volumes of the diluted rmRenin and Trypsin.
  4. Incubate at 37 °C for 1 hour.
  2. Stop Trypsin activity with 1 mM AEBSF.
  1. Dilute AEBSF to 2 mM in Assay Buffer.
  2. Add a volume of 2 mM AEBSF equal to the reaction volume for 1 mM AEBSF and 50 µg/mL rmRenin.
  3. Incubate at room temperature for 30 minutes.
  3. Dilute Substrate to 4 µM in Assay Buffer.
  4. Dilute rmRenin to 2 ng/µL in Assay Buffer.
  5. Load into a black well plate 50 µL of 2 ng/µL rmRenin (100 ng/well) and start the reaction by adding 50 µL of 4 µM Substrate.
  6. Read at excitation and emission wavelengths of 490 nm and 520 nm (top read), respectively, in kinetic mode for 5 minutes.
  7. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank

  **Derived using calibration standard calibration standard 5-FAM (AnaSpec, Catalog # 60584).

  • rmRenin: 0.1 µg
  • Substrate: 2 µM

Notes

QXL is a trademark of AnaSpec, Inc.

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse Renin Protein, CF

• angiotensin-forming enzyme
• Angiotensinogenase
• EC 3.4.23
• EC 3.4.23.15
• FLJ10761
• HNFJ2
• Prorenin
• REN
• renin precursor, renal
• Renin

Background

Renin is a aspartyl protease that plays a crucial role in the regulation of blood pressure and salt balance through the renin-angiotensin system (1). It cleaves angiotensinogen to generate angiotensin I, which can be further converted by angiotensin converting enzyme (ACE) to angiotensin II. Angiotensin II is the active molecule of the renin-angiotensin system that acts by binding to angiotensin receptors type 1 and 2 (AT-1 and AT-2), and has direct pathophysiological effects on the heart and peripheral vasculature (2). After secretion, inactive prorenin can be proteolytically activated by trypsin, cathepsin B, or other proteinases. Because of its unique specificity, inhibition of Renin is widely studied to provide selective therapy for hypertension, congestive heart failure and associated disorders (3).

1. Fuminaki, S. et al. 2004 in Handbook of Proteolytic Enzymes, Barrett, A. J. et al. eds:54.
2. Unger, T. et al. 2008 Medscape J. Med. 10:S4.
3. Dhanaraj, V. et al. 1992 Nature 357:466.

Bioinformatics

• Gene Symbol: Ren1
• Uniprot:
  • P06281()