Legumain Protein, Human (HEK293, C-His)

Legumain proteins can slowly cleave aspartyl bonds, especially under acidic conditions.Legumain Protein, Human (HEK293, C-His) is the recombinant human-derived Legumain protein, expressed by HEK293 , with C-6*His labeled tag.

Legumain protein exhibits a strict specificity for the hydrolysis of asparaginyl bonds. Additionally, it demonstrates the ability to cleave aspartyl bonds slowly, particularly in acidic conditions, further expanding its enzymatic versatility. Functionally, Legumain is integral to the processing of proteins for MHC class II antigen presentation within the lysosomal/endosomal system. It also plays a crucial role in MHC class I antigen presentation in cross-presenting dendritic cells by facilitating the cleavage and maturation of Perforin-2 (MPEG1), thereby promoting antigen translocation in the cytosol, as indicated by recent research findings. Moreover, Legumain is essential for normal lysosomal protein degradation in renal proximal tubules and is required for the degradation of internalized EGFR, highlighting its importance in cellular processes and the regulation of cell proliferation.

Measured by its ability to cleave the fluorogenic peptide substrate, Z-Ala-Ala-Asn-AMC (HY-136626). The specific activity is >250 pmol/min/µg. (Activation description: The proenzyme needs to be activated in acid buffer for an activated form.)

Materials
Activation buffer: 0.1 M NaOAc, 0.1 M NaCl, pH 4.0
Assay buffer: 50 mM MES, 250 mM NaCl, pH 5.0
Test protein: Legumain Protein, Human (HEK293, C-His) (HY-P70897A)
Substrate: Z-Ala-Ala-Asn-AMC (HY-136626) , dissolved in DMSO to a concentration of 5 mM.
Standard: 7-amino, 4-Methyl Coumarin (AMC, HY-137784）

Procedure
1. Dilute AMC in assay buffer to concentrations of 0, 6.25, 12.5, 25, 50, and 100 μmol/L. Add 100 μL of each dilution to a black microplate well.
2. Measure fluorescence at excitation/emission wavelengths of 380 nm/460 nm in kinetic mode for 5 minutes.
3. Plot the measured RFU values on the y-axis and the standard concentrations on the x-axis to generate a standard curve and obtain the curve equation.
4. Dilute the protein to 100 μg/mL in activation buffer and incubate at 37°C for 2 hours.
5. Dilute the protein solution to 10 ng/μL in detection buffer.
6. Dilute the substrate to 200 μM in assay buffer.
7. Add 50 μL of 10 ng/μL Human Legumain to the wells, followed by 50 μL of 200 μM substrate to initiate the reaction.
8. Read in kinetic mode for 5 minutes at excitation and emission wavelengths of 380 nm and 460 nm, respectively.
9. Calculate specific activity: