Recombinant Human LEKTI/SPINK5 Protein, CF

R&D Systems | Catalog # 8564-PI

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Key Product Details

  • R&D Systems HEK293-derived Recombinant Human LEKTI/SPINK5 Protein (8564-PI)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

HEK293

Accession Number

Q9NQ38

Applications

Enzyme Activity
View all LEKTI/SPINK5 Proteins and Enzymes »

Product Specifications

Source

Human embryonic kidney cell, HEK293-derived human LEKTI/SPINK5 protein
Glu626-Glu1064, with a C-terminal 6-His tag

Purity

>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Endotoxin Level

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

N-terminal Sequence Analysis

Glu626

Predicted Molecular Mass

51 kDa

SDS-PAGE

57-65 kDa, reducing conditions

Activity

Measured by its ability to inhibit Recombinant Human Kallikrein 7 (Catalog # 2624-SE) cleavage of a fluorogenic peptide substrate Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (Catalog # ES002).
The IC50 value is <70 nM, as measured under the described conditions.

Formulation, Preparation, and Storage

8564-PI
Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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.

Background: LEKTI/SPINK5

LEKTI, encoded by the SPINK5 gene, is a 145 kDa glycosylated serine proteinase inhibitor that contains 15 Kazal-like domains (D1-15) (1). Mutations in SPINK5 and loss of its expression are associated with the development of various skin disorders such as Netherton syndrome, atopic eczema, and atopic dermatitis (2-4). Alternative splicing of human LEKTI generates a 148 kDa isoform with an insertion between D13 and D14, and a 125 kDa isoform which is truncated following D13 (4, 5). Intracellular Furin-mediated cleavage of LEKTI isoforms between domains D9 and D10 generates multiple secreted fragments (4, 6, 7). Within domains D10-15, human LEKTI shares 61% and 63% aa sequence identity with mouse and rat LEKTI, respectively. Additional LEKTI-derived peptides are found in blood ultrafiltrate (1). LEKTI is primarily expressed in differentiated keratinocytes (4, 8) but is also expressed in the pituitary (9). LEKTI and its fragments can inhibit a range of serine proteases including Kallikreins, Plasmin, Subtilisin A, Cathepsin G, Elastase, and Trypsin (9, 10). Of particular importance in the skin, LEKTI regulates the ability of KLK5, KLK7, and KLK14 to degrade the desmosomal proteins Desmoglein-1 and Desmoplakin-1 during desquamation (3, 6, 7, 11). The D10-15 fragment can inhibit both KLK5 and KLK7 (11) and in turn can be degraded by KLK7 (7). Trypsin-1/PRSS1 promotes KLK5 and KLK7 activity by cleaving the zymogen forms of those proteases and also by degrading multiple LEKTI fragments, including D10-15 (11).

References

  1. Mager, H.-J. et al. (1999) J. Biol. Chem. 274:21499.
  2. Chavanas, S. et al. (2000) Nat. Genet. 25:141.
  3. Descargues, P. et al. (2005) Nat. Genet. 37:56.
  4. Bitoun, E. et al. (2003) Hum. Mol. Genet. 12:2417.
  5. Tartaglia-Polcini, A. et al. (2005) J. Invest. Dermatol. 126:315.
  6. Deraison, C. et al. (2007) Mol. Biol. Cell 18:3607.
  7. Fortugno, P. et al. (2011) J. Invest. Dermatol. 131:2223.
  8. Ishida-Yamamoto, A. et al. (2005) J. Invest. Dermatol. 12:360.
  9. Komatsu, N. et al. (2007) Clin. Chim. Acta 377:228.
  10. Mitsudo, K. et al. (2003) Biochemistry 42:3874.
  11. Miyai, M. et al. (2014) J. Invest. Dermatol.134:1665.

Long Name

Serine Peptidase Inhibitor, Kazal Type 5

Alternate Names

LETKI, NETS, SPINK5, VAKTI

Entrez Gene IDs

11005 (Human); 72432 (Mouse); 361319 (Rat)

Gene Symbol

SPINK5

UniProt

Q9NQ38

Additional LEKTI/SPINK5 Products

Product Documents for Recombinant Human LEKTI/SPINK5 Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human LEKTI/SPINK5 Protein, CF

For research use only

Citations for Recombinant Human LEKTI/SPINK5 Protein, CF

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Protocols

View specific protocols for Recombinant Human LEKTI/SPINK5 Protein, CF (8564-PI):

Materials
  • Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (v/v) Brij-35, pH 7.5 (TCNB)
  • Assay Buffer: 50 mM Tris, 150 mM NaCl, pH 8.5
  • Recombinant Human LEKTI/SPINK5 (rhLEKTI) (Catalog # 8564-PI)
  • Recombinant Human Kallikrein 7 (rhKLK7) (Catalog # 2624-SE)
  • Bacterial Thermolysin (Catalog # 3097-ZN)
  • EDTA (Sigma, Catalog # E-4884)
  • Substrate: Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (Catalog # ES002)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhKLK7 to 200 μg/mL in Activation Buffer.
  2. Dilute Thermolysin to 20 μg/mL in Activation Buffer.
  3. Combine equal volumes of 200 μg/mL rhKLK7 with 20 μg/mL Thermolysin.
  4. Incubate at 37 ˚C for 2 hours.
  5. Stop reaction by adding EDTA to a final concentration of 5 mM.
  6. Dilute incubated rhKLK7 to 8 ng/μL in Assay Buffer.
  7. Prepare a curve of rhLEKTI (MW: 50627 Da) in Assay Buffer by diluting to 1300 nM followed by seven one-half serial  dilutions.
  8. Combine equal volumes of 8 ng/μL rhKLK7 with rhLEKTI serial curve dilutions. Include two controls containing 8 ng/μL rhKLK7 with Assay Buffer.
  9. Incubate reaction mixtures at room temperature for 30 minutes.
  10. Dilute Substrate to 20 µM in Assay Buffer.
  11. Load 50 µL of the diluted reaction mixtures in a plate, and start the reactions by adding 50 µL of 20 µM Substrate to wells.
  12. Read at excitation and emission wavelengths of 320 nM and 405 nM (top read), respectively, in kinetic mode for 5 minutes.
  13. Derive the 50% inhibiting concentration (IC50) value for rhLEKTI by plotting RFU/min (or specific activity) versus  concentration with 4-PL fitting.
  14. Calculate specific activity for rhKLK7 at each point using the following formula (if needed):

     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 MCA-P-L-OH (Bachem, Catalog # M-1975).

  • rhKLK7: 0.2 μg
  • rhLEKTI curve: 325, 162.5, 81.25, 40.625, 20.313, 10.156, 5.078, and 2.539 nM
  • Substrate: 10 µM

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