CTRB1 Protein, Human (HEK293, His)

Chymotrypsinogen B (CTRB1) is the precursor of the pancreatic proteolytic enzymes. CTRB1 is synthesized in the acinar cells of the pancreas and secreted into the small intestine where it undergoes proteolytic activation to generate the functional enzyme. CTRB1 enables serine-type endopeptidase activity, being involved in response to apoptotic signal, nutrient, cytokine and peptide hormone. CTRB1 is located in lysosome and is a biomarker of pancreatitis. CTRB1 Protein, Human (HEK293, His) is the recombinant human-derived CTRB1 protein, expressed by HEK293 , with C-6*His labeled tag.

Chymotrypsinogen B (CTRB1) is a member of the serine protease family of enzymes and forms a principal precursor of the pancreatic proteolytic enzymes. CTRB1 is synthesized in the acinar cells of the pancreas and secreted into the small intestine where it undergoes proteolytic activation to generate the functional enzyme. CTRB1 enables serine-type endopeptidase activity, being involved in response to apoptotic signal, nutrient, cytokine and peptide hormone. CTRB1 induces apoptosis through Bid cleavage activity, as the truncated Bid can in turn cause rapid mitochondrial release of cytochrome c. CTRB1 is located in lysosome and is a biomarker of pancreatitis. CTRB1 gene encodes distinct isoforms, which may undergo similar processing to generate the mature protein^{[1][2][3][4][5]}.

Materials
Assay buffer: 0.1 M Tris-HCl, 1 mM CaCl₂, 0.05% TWEEN 20, pH 8.0
Test protein: CTRB1 protein, human (HEK293, His) (HY-P70005)
Substrate: Suc-AAPF-pNA
Standard: p-Nitroaniline

Procedure
1. Dilute p-nitroaniline (standard) with assay buffer to concentrations of 0, 1.171875, 2.34375, 4.6875, 9.375, 18.75, 37.5, 75, 150, and 300 μM.
2. Add 100 µL of each concentration of the standard to the wells.
3. Measure the OD₄₁₀ values using a microplate reader.
4. Plot a standard curve with the molarity of the standard on the y-axis and the measured OD on the x-axis to obtain the standard curve equation.
5. Dilute the substrate to 500 µM using the assay buffer.
6. Add 50 µL of 100 µg/mL human CTRB1 at different concentrations to the wells, then add 50 µL of 500 µM substrate to initiate the reaction. For the control group, add 50 µL of assay buffer and 50 µL of 500 µM substrate.
7. On a microplate reader, read for 5 minutes in kinetic mode at an OD₄₁₀ wavelength.
8. Calculate the specific activity:

Human

HEK293

C-6*His

P17538 (C19-N263)

1504  [NCBI]

Full Length of Mature Protein

CGVPAIHPVLSGLSRIVNGEDAVPGSWPWQVSLQDKTGFHFCGGSLISEDWVVTAAHCGVRTSDVVVAGEFDQGSDEENIQVLKIAKVFKNPKFSILTVNNDITLLKLATPARFSQTVSAVCLPSADDDFPAGTLCATTGWGKTKYNANKTPDKLQQAALPLLSNAECKKSWGRRITDVMICAGASGVSSCMGDSGGPLVCQKDGAWTLVGIVSWGSDTCSTSSPGVYARVTKLIPWVQKILAAN

28-30 kDa

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

<1 EU/μg, determined by LAL method.