Recombinant Human Neprilysin (carrier-free)

Product Details

Source

Human, amino acid (Tyr52-Trp750) (Accession: NM_000789.4), with a N-terminal 6His-GGS tag was expressed in 293E cell.

Molecular Mass

The 708 amino acid recombinant protein has a predicted molecular mass of approximately 80.8 kD. The DTT-reduced and non-reduced protein migrates at approximately 80 kD by SDS-PAGE. The predicted N-terminal amino acid is His.

Purity

> 95%, as determined by Coomassie stained SDS-PAGE

Formulation

Tris pH 7.2, 50 mM NaCl, 5% glycerol

Concentration

10 and 25 µg sizes are bottled at 200 µg/mL. 100 µg size and larger sizes are lot-specific and bottled at the concentration indicated on the vial. To obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.

Storage & Handling

Unopened vial can be stored at -20°C or -70°C for six months. For maximum results, quick spin vial prior to opening. Avoid repeated freeze/thaw cycles.

Activity

Human Neprilysin activity is measured by its ability to cleave the fluorogenic peptide substrate MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH. The specific activity is > 1500 pmol/min/µg in the presence of 0.005 µg of recombinant human Neprilysin.

Application

Bioassay

Application Notes

Human Neprilysin Enzymatic Assay

Human Neprilysin (CD10) activity is measured by its ability to cleave the fluorogenic peptide substrate MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH. The increase of the product is monitored by an increase in intensity of fluorescence at 405 nm with excitation at 320 nm. Perform the assay rapidly. The specific activity is > 1500 pmol/min/µg in the presence of 0.005 µg of recombinant human Neprilysin.


Materials

• Assay Buffer: 50 mM Tris, 0.05% Brij-35, pH 9.0
• Human Neprilysin
• Fluorogenic peptide substrate: MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH
• F16 Black Maxisorp Plate (Nunc, Cat. No. 475515)

Activity Assay Procedure

1. Dilute the substrate in assay buffer at 20 µM. Wrap the substrate container in aluminium foil.
2. In assay buffer, prepare 50 µL of serial dilutions of human Neprilysin. Start at a concentration of 3.2 µg/mL and dilute 1 to 2 ten times after.
3. Add 50 µL of 20 µM substrate to wells with 50 µL of prepared dilutions.
4. Include a substrate blank with 50 µL of assay buffer and 50 µL of 20 µM substrate without any human Neprilysin.
5. Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes.
6. Calculate specific activity using the following formula:

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

 

Final assay conditions per well:
Human Neprilysin: 0.16, 0.08, 0.04, 0.02, 0.01, 0.005, 0.0025, 0.00125, 0.000625, 0.0003125, 0.00015625 µg.
Substrate: 10 µM

BioLegend carrier-free recombinant proteins provided in liquid format are shipped on blue ice. Our comparison testing data indicates that when handled and stored as recommended, the liquid format has equal or better stability and shelf-life compared to commercially available lyophilized proteins after reconstitution. Our liquid proteins are verified in-house to maintain activity after shipping on blue ice and are backed by our 100% satisfaction guarantee. If you have any concerns, contact us at tech@biolegend.com.

Antigen Details

Structure

Monomer

Distribution

Leukemic cells, pre B cells, germinal centers B cells, granulocytes, and fibroblasts. Abundant expression in duodenum, kidney, fat, and small intestine.  

Function

Inactivates peptide hormones such as bradykinin, substance P, neurotensin, enkephalins, and glucagon.

Interaction

Bradykinin, natriuretic peptides, and adrenomedullin, glucagon, enkephalins, substance P, neurotensin, angiotensin-1, angiotensin-2, and oxytocin.

Bioactivity

Human Neprilysin cleaves a fluorogenic peptide substrate MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH. The specific activity is > 1500 pmol/min/µg in the presence of 0.005 µg of recombinant human Neprilysin.

Cell Sources

293E cell

Cell Type

B cells, Endothelial cells, Epithelial cells, Fibroblasts, Leukemia

Biology Area

Cancer Biomarkers, Cardiovascular Biology, Cell Biology, Inhibitory Molecules, Innate Immunity, Neuroscience

Molecular Family

Enzymes and Regulators, Proteases

Antigen References

1. N Iwata, et al., 2001. Science. 292(5521):1550.
2. S Zraika, et al., 2013. Diabetes. 62:1593.
3. C Bavishi, et al. 2015. Eur. Heart J. 36:1967.
4. E Riddell, and JM Vader. 2017. Curr. Heart Fail. Rep. 14:134.
5. DJ Campbell. 2018. Front Med. 5:257.

Gene ID

4311 View all products for this Gene ID

UniProt

View information about Neprilysin on UniProt.org

Documentation

• Technical Data Sheet (pdf)
• Certificate of Analysis
• Safety Data Sheet

Reviews

Related Pages & Pathways

Related FAQs

Why choose BioLegend recombinant proteins?

     • Each lot of product is quality-tested for bioactivity as indicated on the data sheet.
     • Greater than 95% Purity or higher, tested on every lot of product.
     • 100% Satisfaction Guarantee for quality performance, stability, and consistency.
     • Ready-to-use liquid format saves time and reduces challenges associated with reconstitution.
     • Bulk and customization available. Contact us.
     • Learn more about our Recombinant Proteins.

How does the activity of your recombinant proteins compare to competitors?

We quality control each and every lot of recombinant protein. Not only do we check its bioactivity, but we also compare it against other commercially available recombinant proteins. We make sure each recombinant protein’s activity is at least as good as or better than the competition’s. In order to provide you with the best possible product, we ensure that our testing process is rigorous and thorough. If you’re curious and eager to make the switch to BioLegend recombinants, contact your sales representative today!

What is the specific activity or ED50 of my recombinant protein?

The specific activity range of the protein is indicated on the product datasheets. Because the exact activity values on a per unit basis can largely fluctuate depending on a number of factors, including the nature of the assay, cell density, age of cells/passage number, culture media used, and end user technique, the specific activity is best defined as a range and we guarantee the specific activity of all our lots will be within the range indicated on the datasheet. Please note this only applies to recombinants labeled for use in bioassays. ELISA standard recombinant proteins are not recommended for bioassay usage as they are not tested for these applications.

Have your recombinants been tested for stability?

Our testing shows that the recombinant proteins are able to withstand room temperature for a week without losing activity. In addition the recombinant proteins were also found to withstand four cycles of freeze and thaw without losing activity.

Does specific activity of a recombinant protein vary between lots?

Specific activity will vary for each lot and for the type of experiment that is done to validate it, but all passed lots will have activity within the established ED50 range for the product and we guarantee that our products will have lot-to-lot consistency. Please conduct an experiment-specific validation to find the optimal ED50 for your system.

How do you convert activity as an ED50 in ng/ml to a specific activity in Units/mg?

Use formula Specific activity (Units/mg) = 10^6/ ED50 (ng/mL)

Certificate of Analysis