Recombinant Human DPEP1 His-tag Protein, CF Summary
Details of Functionality |
Measured by its ability to cleave carnosine ( beta -Ala-L-His) in a two-step assay. The specific activity is >5 pmol/min/μg, as measured under the described conditions. |
Source |
Chinese Hamster Ovary cell line, CHO-derived human DPEP1 protein Asp17-Ser384, with C-terminal 6-His tag |
Accession # |
|
N-terminal Sequence |
Asp17 |
Protein/Peptide Type |
Recombinant Enzymes |
Purity |
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining. |
Endotoxin Note |
<0.10 EU per 1 μg of the protein by the LAL method. |
Applications/Dilutions
Dilutions |
|
Theoretical MW |
42 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 |
39-49 kDa, under 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 Tris and NaCl. |
Purity |
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining. |
Assay Procedure |
- Assay Buffer: 50 mM Tris, pH 7.5
- Recombinant Human DPEP1 (rhDEP1) (Catalog # 11524-DP)
- Substrate: L-Carnosine, 100 mM stock in deionized water
- Trichloroacetic acid (TCA), 10% stock in deionized water
- o-Phthaldialdehyde (OPA), 50 mg/mL stock in DMSO
- Sodium Hydroxide (NaOH), 2 M stock in deionized water
- Black 96-well plate
- Plate Reader with Fluorescence Read Capability
- Dilute rhDPEP1 to 20 µg/mL in Assay Buffer.
- Dilute Substrate to 4 mM in Assay Buffer.
- Dilute 10% TCA to 1% in Assay Buffer (prepare right before adding to Substrate Blank(s) in step 4).
- Mix 60 μL of 20 µg/mL rhDPEP1 and 60 μL 4 mM Substrate for a final concentration of 10 µg/mL and 2 mM respectively. Include a Substrate Blank containing 60 μL of 20 µg/mL rhDPEP1, 60 μL 1% TCA added 1 minute prior to substrate addition, and 60 μL Substrate.
- Incubate reactions for 1 hour at 37 °C.
- Dilute 10% TCA to 1% in Assay Buffer (prepare right before adding to reactions in step 7).
- Stop reactions by adding 60 μL of 1% TCA (do not add to Substrate Blank(s)) and incubate for 10 minutes at room temperature.
- Dilute OPA to 5 mg/mL in 2 M NaOH.
- Add 60 μL of 5 mg/mL OPA to each reaction.
- Incubate for 1 hour at room temperature.
- Load 200 µL of each incubated sample into the plate.
- Read at excitation and emission wavelengths of 360 nm and 460 nm (top read), respectively, in endpoint mode.
- Calculate Specific Activity:
Specific Activity (pmol/min/µg) = | Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU) | Incubation time (min) x amount of enzyme (µg) |
*Adjusted for Substrate Blank **Derived using calibration standard L-Histidine. Per Well: - rhDPEP1: 1.0 µg
- Substrate: 1 mM
|
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human DPEP1 His-tag Protein, CF
Background
Dipeptidase 1 (DPEP1), also referred to as renal peptidase or beta-lactamase, is a zinc-dependent, glycosylated member of the M19 peptidase metallo-dependent hydrolase superfamily that lacks a sequence commonly found in zinc proteases and has poor homology to other metallopeptiases (1). DPEP1 is a homodimeric elongated ellipsoid with a single disulfide bond between the subunits (1) and GPI-anchored to the cell membrane at the brush border of epithelial cells (2). Each DPEP1 monomer contains a zinc-dependent active site, a propeptide, and a GPI anchor at the c-terminus (1). DPEP1 has activity towards several dipeptides and is critical in glutathione and leukotriene metabolism as well as mammalian metabolism of the B-lactam ring of antibiotics (1, 3). DPEP1 expression is upregulated or associated as a marker in various cancers including colon, kidney, and prostate cancer (4-6). It is also upregulated in renal tubular epithelial cells and associated with iron-related ferroptotic cell death in diabetic retinopathy (2, 7) where it may mediate proinflammatory cancer mediatory leukotrienes or serve as a receptor as an adhesion receptor for neutrophil recruitment and impact inflammation (8-10). DPEP1 has been noted as a potential therapeutic marker or target for mestastasis, cancer progression, and disease in the liver and lungs with acute or chronic inflammation (2, 4, 9, 11).
- Nitanai, Y. et. al. (2002) J. Mol. Biol. 321:177.
- Li, Y. et. al. (2024) Int. Immunopharmacol. 133:111955.
- Campbell, B.J. et. al. (1988) Biochim. Biophys. Acta 956:110.
- Tachibana, K. et. al. (2017) Biomed. Rep. 6:423.
- Xiang, C. et. al. (2023) J. Cell Mol. Med. 27:1947.
- Zhu, W. et. al. (2023) Heliyon 9:e18870.
- Allison, S.J. (2021) Nat. Rev. Nephrol. 17:707.
- Satoh, S. et. al. (1993) Biochim. Biophys. Acta 1172:181.
- Choudhury, S.R. et. al. (2019) Cell 178:1205.
- Wang, M. (2022) Nat. Rev. Nephrol. 18:199.
- Tutanov. O.S. et. al. (2023) Extracell. Vesicles Circ. Nucl. Acids. 4:195.
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