Cathepsin L1 Protein, Human (HEK293, His, solution)

Cathepsin L1 Protein, Human (HEK293, His, solution) is a major cysteine protease that plays a major role in intracellular protein catabolism.

Cathepsin L1, a major cysteine protease secreted by the parasite plays a pivotal role in various aspects of its pathogenicity. For example, Cathepsin L1 takes part in nutrient acquisition by catabolizing host proteins to absorbable peptides, facilitates the migration of the parasite through the host intestine and liver by cleaving interstitial matrix proteins such as fibronectin, laminin, and native collagen, and is implicated in the inactivation of host immune defenses by cleaving immunoglobulins. Cathepsin L1 suppresses Th1 immune responses in infected laboratory animals making them susceptible to concurrent bacterial infections^[1].

1.Biological activity has been tested in vitro.
2.Measured by its ability to cleave 80μM fluorogenic peptide substrate Z-LR-AMC that incubate at room temperature in kinetic mode for 5 minutes. The specific activity is 282826.08 pmol/min/µg.(Activation description: The proenzyme needs to be activated on ice for an activated form.)

Materials
Assay buffer: 50 mM MES, 5 mM DTT, 1 mM EDTA, 0.005% (w/v) Brij-35, pH 6.0
Test protein: Cathepsin L1 Protein, Human (HEK293, His, solution) (HY-P7754)
Substrate: Z-Leu-Arg-AMC (HY-142021)
Standard: 7-Amino-4-methylcoumarin (AMC, HY-D0027)

Procedure
1. Standard Curve: Dilute AMC to concentrations of 100, 50, 25, 12.5, 6.25, and 0 μM using assay buffer. Add 50 μL of each dilution to black microplate wells. Read for 5 minutes in kinetic mode at excitation and emission wavelengths of 380 nm and 460 nm respectively. Plot the standard curve with the measured RFU values on the x-axis and the molar quantity of the standard substance on the y-axis to derive the standard curve equation.
2. Dilute Human Cathepsin L1 to 40 μg/mL using the assay buffer. 3. Incubate the diluted Human Cathepsin L1 on ice for 15 minutes. 4. Dilute Human Cathepsin L1 to 0.02 μg/mL using assay buffer. 5. Dilute the substrate to 80 μM using assay buffer. 6. Add different concentrations of Human Cathepsin L1 to a black microplate, 50 μL per well, and initiate the reaction by adding 50 μL of 80 μM substrate. Add 50 μL assay buffer and 50 μL 80 μM substrate to blank wells. 7. Read in kinetic mode at excitation wavelength 380 nm and emission wavelength 460 nm for 5 minutes.
8. Calculate specific activity:

Human

HEK293

C-6*His

P07711-1 (T18-V333)

1514  [NCBI]

Full Length of Mature Protein

TLTFDHSLEAQWTKWKAMHNRLYGMNEEGWRRAVWEKNMKMIELHNQEYREGKHSFTMAMNAFGDMTSEEFRQVMNGFQNRKPRKGKVFQEPLFYEAPRSVDWREKGYVTPVKNQGQCGSCWAFSATGALEGQMFRKTGRLISLSEQNLVDCSGPQGNEGCNGGLMDYAFQYVQDNGGLDSEESYPYEATEESCKYNPKYSVANDTGFVDIPKQEKALMKAVATVGPISVAIDAGHESFLFYKEGIYFEPDCSSEDMDHGVLVVGYGFESTESDNNKYWLVKNSWGEEWGMGGYVKMAKDRRNHCGIASAASYPTV

Approximately 34 kDa, based on SDS-PAGE under reducing conditions.

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

Solution.

Supplied as a 0.2 μm filter solution of 20 mM HAc-NaAc, 150 mM NaCl, pH 4.5.

<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.

• [1]. Panat Anuracpreeda, et al. Production and characterization of a monoclonal antibody against recombinant cathepsin L1 of Fasciola gigantica. Acta Trop. 2014 Aug;136:1-9.  [Content Brief]