ELANE Protein, Human (GST)

ELANE is a serine protease that critically regulates natural killer cells, monocytes, and granulocytes by inhibiting C5a-dependent neutrophil enzyme release and chemotaxis, thereby shaping immune responses. It also inhibits pyroptosis by cleaving GSDMB, affecting the regulation of programmed cell death. ELANE Protein, Human (GST) is the recombinant human-derived ELANE protein, expressed by E. coli , with N-GST labeled tag.

ELANE, a serine protease, plays a crucial role in modulating the functions of natural killer cells, monocytes, and granulocytes. It acts by inhibiting C5a-dependent neutrophil enzyme release and chemotaxis, highlighting its regulatory role in immune responses. Additionally, ELANE participates in the inhibition of pyroptosis by promoting the cleavage of GSDMB, showcasing its involvement in the regulation of programmed cell death. Notably, ELANE exhibits bactericidal activity by effectively killing E. coli in vitro, while sparing S. aureus. This antimicrobial effect is attributed to its ability to digest outer membrane protein A (ompA) in E. coli and K. pneumoniae, underscoring its role in bacterial defense mechanisms.

Measured by its ability to cleave the fluorogenic peptide substrate, MeOSuc-Ala-Ala-Pro-Val-7-amido-4-methylcoumarin (MeOSuc-AAPV-AMC). The specific activity is 1776.266 pmol/min/µg, as measured under the described conditions. (Activation description: The proenzyme needs to be activated by Mouse Cathepsin C (HY-P704018) for an activated form.)

Materials
Activation buffer: 50 mM MES, 50 mM NaCl, pH 5.5
Assay buffer: 50 mM Tris, 1 M NaCl, 0.05% (w/v) Brij-35, pH 7.5
Test protein: ELANE Protein, Human (GST)(HY-P72179)
Activator protein: Cathepsin C/DPPI Protein, Mouse (Active, HEK293, His) (HY-P704018)
Substrate: Lys-Pro-AMC diTFA (HY-P4426A)
Standard: 7-amino, 4-Methly Coumarin (HY-D0027)

Procedure
1. Dilute Human ELANE to 50 μg/mL in activation buffer containing 50 μg/mL Mouse Cathepsin C/DPP1.
2. Incubate at 37°C for 2 hours to activate Human ELANE.
3. Dilute the activated Human ELANE to 1 ng/μL in assay buffer.
4. Dilute the substrate to 200 μM in assay buffer.
5. Load 50 μL of 1 ng/μL Human ELANE onto the plate and initiate the reaction by adding 50 μL of 200 μM substrate. Include a substrate blank comprising 50 μL assay buffer and 50 μL of 200 μM substrate.
6. In kinetic mode, read at excitation wavelength 380 nm and emission wavelength 460 nm (top reading) for 5 minutes.
7. Dilute 7-amino, 4-Methyl Coumarin to concentrations of 0, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μM using assay buffer.
8. Add 100 μL of each standard concentration to the wells.
9. In kinetic mode, read at excitation wavelength 380 nm and emission wavelength 460 nm (top-reading) for 5 minutes.
10. Plot a standard curve with standard concentration on the y-axis and RFU values on the x-axis to derive the standard curve equation.
11. Calculate Specific Activity:

Human

E. coli

N-GST

P08246 (I30-H267)

1991  [NCBI]

Full Length of Mature Protein

IVGGRRARPHAWPFMVSLQLRGGHFCGATLIAPNFVMSAAHCVANVNVRAVRVVLGAHNLSRREPTRQVFAVQRIFENGYDPVNLLNDIVILQLNGSATINANVQVAQLPAQGRRLGNGVQCLAMGWGLLGRNRGIASVLQELNVTVVTSLCRRSNVCTLVRGRQAGVCFGDSGSPLVCNGLIHGIASFVRGGCASGLYPDAFAPVAQFVNWIDSIIQRSEDNPCPHPRDPDPASRTH

Approximately 52.6 kDa

≥ 90%, as determined by reducing SDS-PAGE.

Lyophilized powder.

Lyophilized from a 0.22 μm filtered solution of 10 mM Tris-HCl, 1 mM EDTA, 6% trehalose, pH 8.0.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O.

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.