Recombinant Human LEKTI/SPINK5 Protein, CF Summary
Product Specifications
Glu626-Glu1064, with a C-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
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.
|
Assay Procedure
- 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
- Dilute rhKLK7 to 200 μg/mL in Activation Buffer.
- Dilute Thermolysin to 20 μg/mL in Activation Buffer.
- Combine equal volumes of 200 μg/mL rhKLK7 with 20 μg/mL Thermolysin.
- Incubate at 37 ˚C for 2 hours.
- Stop reaction by adding EDTA to a final concentration of 5 mM.
- Dilute incubated rhKLK7 to 8 ng/μL in Assay Buffer.
- Prepare a curve of rhLEKTI (MW: 50627 Da) in Assay Buffer by diluting to 1300 nM followed by seven one-half serial dilutions.
- Combine equal volumes of 8 ng/μL rhKLK7 with rhLEKTI serial curve dilutions. Include two controls containing 8 ng/μL rhKLK7 with Assay Buffer.
- Incubate reaction mixtures at room temperature for 30 minutes.
- Dilute Substrate to 20 µM in Assay Buffer.
- 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.
- Read at excitation and emission wavelengths of 320 nM and 405 nM (top read), respectively, in kinetic mode for 5 minutes.
- Derive the 50% inhibiting concentration (IC50) value for rhLEKTI by plotting RFU/min (or specific activity) versus concentration with 4-PL fitting.
- 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
Reconstitution Calculator
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).
- Mager, H.-J. et al. (1999) J. Biol. Chem. 274:21499.
- Chavanas, S. et al. (2000) Nat. Genet. 25:141.
- Descargues, P. et al. (2005) Nat. Genet. 37:56.
- Bitoun, E. et al. (2003) Hum. Mol. Genet. 12:2417.
- Tartaglia-Polcini, A. et al. (2005) J. Invest. Dermatol. 126:315.
- Deraison, C. et al. (2007) Mol. Biol. Cell 18:3607.
- Fortugno, P. et al. (2011) J. Invest. Dermatol. 131:2223.
- Ishida-Yamamoto, A. et al. (2005) J. Invest. Dermatol. 12:360.
- Komatsu, N. et al. (2007) Clin. Chim. Acta 377:228.
- Mitsudo, K. et al. (2003) Biochemistry 42:3874.
- Miyai, M. et al. (2014) J. Invest. Dermatol.134:1665.
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