Kallikrein-2 Protein, Human (HEK293, His)

The Kallikrein-2 protein is a member of the glandular kallikrein family and is critical in the enzymatic process as it selectively cleaves the Met-Lys and Arg-Ser bonds in kininogen, thereby releasing Lys-bradykinin . This proteolytic activity contributes to the production of bradykinin, a bioactive peptide with multiple roles in regulating blood pressure, inflammation, and vascular permeability. Kallikrein-2 Protein, Human (HEK293, His) is the recombinant human-derived Kallikrein-2 protein, expressed by HEK293 , with C-6*His labeled tag.

Kallikrein-2 is a member of the glandular kallikrein family, exhibiting enzymatic activity by cleaving Met-Lys and Arg-Ser bonds in kininogen, resulting in the release of Lys-bradykinin. As part of the kinin-kallikrein system, Kallikrein-2 contributes to the generation of bradykinin, a potent vasoactive peptide involved in the regulation of blood pressure, inflammation, and vascular permeability. By specifically cleaving these bonds in kininogen, Kallikrein-2 plays a crucial role in the controlled release of Lys-bradykinin, influencing downstream physiological processes.

Measured by its ability to cleave a flourogenic peptide substrate Z-Phe-Arg-7-amido-4-methylcoumarin (FR-AMC). The specific activity is >125 pmol/min/μg.

Materials
Assay buffer: 50 mM Tris-HCl, 0.15 M NaCl, 10 mM CaCl?, 0.05% (w/v) Brij-35, pH 7.5
Test protein: Kallikrein-2 Protein, Human (HEK293, His)
Substrate: Z-Phe-Arg-AMC, 10 mM stock in DMSO
Standard: AMC, 10 mM stock in DMSO

Procedure
1. Prepare calibration curve: Dilute the standard solution to 3 μM with assay buffer, then perform six serial dilutions. Dispense 100 μL of each dilution into black microplate wells. Read in kinetic mode for 5 minutes at excitation (380 nm) and emission (460 nm) wavelengths (top read). Plot the standard curve with measured RFU on the y-axis and standard substance amount on the x-axis to obtain the standard curve equation.
2. Dilute Human Kallikrein-2 to 5 μg/mL in assay buffer.
3. Dilute substrate Z-Phe-Arg-AMC to 200 μM in assay buffer.
4. Instrument Settings: Select Kinetic and Fluorescence functions. For Kinetic mode, set 11 cycles with 30-second intervals between cycles. For Fluorescence, set excitation at 380 nm and emission at 460 nm. 5. Experimental group: 50 μL, 5 μg/mL protein + 50 μL, 200 μM substrate.
Blank group: 50 μL assay buffer + 50 μL, 200 μM substrate.
6. Reading: Place the microplate into the microplate reader for reading.
7. Calculate specific activity:

Human

HEK293

C-6*His

P20151-1 (P19-P261)

3817  [NCBI]

Full Length of Isoform-1 Mature Protein

PLIQSRIVGGWECEKHSQPWQVAVYSHGWAHCGGVLVHPQWVLTAAHCLKKNSQVWLGRHNLFEPEDTGQRVPVSHSFPHPLYNMSLLKHQSLRPDEDSSHDLMLLRLSEPAKITDVVKVLGLPTQEPALGTTCYASGWGSIEPEEFLRPRSLQCVSLHLLSNDMCARAYSEKVTEFMLCAGLWTGGKDTCGGDSGGPLVCNGVLQGITSWGPEPCALPEKPAVYTKVVHYRKWIKDTIAANP

Approximately (10-12)&(16-19)&(28-33) KDa

≥ 95%, as determined by reducing SDS-PAGE.

Solution

Supplied as a 0.2 μm filtered solution of 20 mM Acetate, 250 mM Trehalose, 0.02% Tween 80, pH 5.0.

<1 EU/μg, determined by LAL method.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.