Cathepsin B Protein, Mouse (HEK293, His)

Cathepsin B Protein, Mouse (HEK293, His) is an approximately 32-50 kDa Cathepsin B protein with a His-flag. Cathepsin B is an enzymatic protein belonging to the peptidase C1 family^[1].

Cathepsin B (CTSB) is also known as APP secretase (APPS) and CPSB, is an enzymatic protein belonging to the peptidase C1 family^[1].
Cathepsin B/CTSB is synthesized as a preproenzyme and is activated in prelysosomal acidic vesicles (late endosomes) prior to its delivery to lysosomes. After removal of the signal peptide, the inactive proenzyme undergoes modifications including removal of the pro region to result in the active enzyme^[1].
Several lysosomal proteinases including the cathepsin B have been implicated in malignant progression of tumors. Many researchers have demonstrated correlations between increased activity of cathepsin B and increased metastatic capability of animal tumors or malignancy of human tumors^[2].

Measured by its ability to cleave 10 μM fluorogenic peptide 10 μM substrate Z-LR-AMC (HY-142021). The specific activity is >2000 pmol/min/µg, as measured under the described conditions. (Activation description: The proenzyme needs to be activated in acid reducing buffer for an activated form.)

Materials
Activation Buffer: 25 mM MES, 5 mM DTT, pH 5.0.
Assay Buffer: 25 mM MES, pH 5.0.
Cathepsin B Protein, Mouse (HEK293, His) (HY-P7747)
Substrate: 7-Amino, 4-Methyl Coumarin (AMC, HY-D0027)

Experimental steps
1. Standard curve: Dilute 7-Amino, 4-Methyl Coumarin (AMC) (standard) to 0, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100 μmol/L with assay buffer, and add 100 μL into the black enzyme-labeled wells. The excitation and emission wavelengths were 380 nm and 460 nm, respectively, and the standard curve was made with the measured RFU as the horizontal coordinate and the amount of standard substance as the vertical coordinate to obtain the standard curve formula.
2. Dilute the measured proteins to 10μg/mL with activation buffer.
3. Incubate for 15 minutes at room temperature.
4. Dilute Mouse Cathepsin B to 0.2 μg/mL in Assay Buffer.
5. Dilute substrate to 20 μM in assay buffer.
6. Add 50 μL of each concentration of the measured protein to the assay wells and add 50 μL of 20 μM substrate to start the reaction. Blank wells were spiked with 50 μL of assay buffer and 50 μL of 20μM protein-free substrate.
7. The excitation and emission wavelengths were 380 nm and 460 nm, respectively, and read in kinetic mode for 5 minutes.
8. Calculate the specific activity:

Mouse

HEK293

C-6*His

P10605 (H18-F339)

13030  [NCBI]

Full Length of Mature Protein

HDKPSFHPLSDDLINYINKQNTTWQAGRNFYNVDISYLKKLCGTVLGGPKLPGRVAFGEDIDLPETFDAREQWSNCPTIGQIRDQGSCGSCWAFGAVEAISDRTCIHTNGRVNVEVSAEDLLTCCGIQCGDGCNGGYPSGAWSFWTKKGLVSGGVYNSHVGCLPYTIPPCEHHVNGSRPPCTGEGDTPRCNKSCEAGYSPSYKEDKHFGYTSYSVSNSVKEIMAEIYKNGPVEGAFTVFSDFLTYKSGVYKHEAGDMMGGHAIRILGWGVENGVPYWLAANSWNLDWGDNGFFKILRGENHCGIESEIVAGIPRTDQYWGRFHHHHHH

Approximately 32-50 kDa, based on SDS-PAGE under reducing conditions, due to the glycosylation.

Greater than 95% as determined by reducing SDS-PAGE.

Lyophilized powder.

Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O. For long term storage it is recommended to add a carrier protein (0.1% BSA, 5% HSA, 10% FBS or 5% Trehalose).

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.

• [1]. Izabela Podgorski, et al. Cathepsin B and its role(s) in cancer progression. Biochem Soc Symp. 2003;(70):263-76.  [Content Brief]

  [2]. W Morikawa, et al. Angiostatin generation by cathepsin D secreted by human prostate carcinoma cells. J Biol Chem. 2000 Dec 8;275(49):38912-20.  [Content Brief]