Cathepsin S Protein, Human (HEK293, His)

Cathepsin S Protein, Human (HEK293, His) is potent cysteine protease which can promote degradation of damaged or unwanted proteins in the endo-lysosomal pathway.

Cathepsin S is a member of the cysteine cathepsin protease family. Cathepsin S has specific roles such as MHC class II antigen presentation, where it is important in the degradation of the invariant chain. Cathepsin S is involved in a variety of pathological processes including arthritis, cancer, and cardiovascular disease, where it becomes secreted and can act on extracellular substrates. Cathepsin S has uniquely restricted tissue expression and is more stable at a neutral pH. Cathepsin S is unique amongst the cysteine cathepsin family due to restricted tissue expression, associated with antigen presenting cells localised in lymph and spleen, as well as other immune cells such as macrophages. Cathepsin S is thought to be a particularly potent cysteine protease cleaving elastin and generating bioactive elastin peptides, leading to the promotion of cardiovascular inflammation and calcification. Cathepsin S is also released by smooth muscle cells and macrophages as a systemic response to inflammation in a continuous recursive feedback loop^[1][2].

Measured by its ability to cleave a fluorogenic peptide substrate, (7-methoxycoumarin-4-yl) acetyl-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys (2, 4-dinitrophenyl)-NH2 (R&D Systems, Catalog#ES002). Cleavage of ES002 can be measured using excitation and emission wavelength at 320 nm and 405 nm, respectively.The specific activity is >300 pmoles/min/μg. (Activation description: The enzyme needs to be activated in acid buffer for an activated form.)

Materials
Assay buffer:50 mM NaOAc, 5 mM DTT, 250 mM NaCl, pH 4.5. Test protein: Cathepsin S Protein, Human (HEK293, His) (HY-P7756)
Substrate: (7-methoxycoumarin-4-yl) acetyl-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys (2, 4-dinitrophenyl)-NH₂
Standard: MCA-Pro-Leu-OH

Procedure
1. Dilute Human Cathepsin S to 100 μg/mL in assay buffer.
2. Incubate at room temperature for 2 hours.
3. Dilute Human Cathepsin S to 2 ng/μL in assay buffer.
4. Dilute Substrate to 20 μM in assay buffer.
5. Load 50 μL of the 2 ng/μL Human Cathepsin S into a black well plate, and start the reaction by adding 50 μL of 20 μM substrate. Include a Substrate Blan k containing 50 μL assay buffer and 50 μL of 20 μM.
Substrate without any rh Cathepsin S.
6. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
7. Prepare a curve of standard MCA-Pro-Leu-OH in assay buffer. Make the following serial dilutions: 4 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM, 0.125 μM, 0.0625 μM mM and 0 μM. Then transfer 100 μL serial standards to the plate.
8. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in endpoint mode.
9. Calculate the specific activity:

Human

HEK293

C-His

NP_004070.3 (Q17-I331)

1520  [NCBI]

Full Length of Mature Protein (with Propeptide)

QLHKDPTLDHHWHLWKKTYGKQYKEKNEEAVRRLIWEKNLKFVMLHNLEHSMGMHSYDLGMNHLGDMTSEEVMSLMSSLRVPSQWQRNITYKSNPNRILPDSVDWREKGCVTEVKYQGSCGACWAFSAVGALEAQLKLKTGKLVSLSAQNLVDCSTEKYGNKGCNGGFMTTAFQYIIDNKGIDSDASYPYKAMDQKCQYDSKYRAATCSKYTELPYGREDVLKEAVANKGPVSVGVDARHPSFFLYRSGVYYEPSCTQNVNHGVLVVGYGDLNGKEYWLVKNSWGHNFGEEGYIRMARNKGNHCGIASFPSYPEI

37 kDa

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

Solution

1. Supplied as a 0.2 μm filter solution of 20 mM MES, 150 mM NaCl, 10% Glycerol, pH 5.5.
2. Supplied as a 0.2 μm filter solution of 12.5 mM MES, 75 mM NaCl, 10% Glycerol, 400 nM Leupeptin, 16 nM Aproteinin, pH 6.5.
3. Supplied as a 0.2 μm filter solution of 12.5 mM MES, 75 mM NaCl, pH 6.5 with 50% glycerol.
Please refer to the lot-specific COA for specific buffer information.

<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]. Richard D A Wilkinson, et al. Cathepsin S: therapeutic, diagnostic, and prognostic potential. Biol Chem. 2015 Aug;396(8):867-82.  [Content Brief]

  [2]. Brena F Sena, et al. Cathepsin S As an Inhibitor of Cardiovascular Inflammation and Calcification in Chronic Kidney Disease. Front Cardiovasc Med. 2018 Jan 5;4:88.  [Content Brief]