Recombinant Human 2/PCSK2 Catalog Number: 6018-SE

DESCRIPTION Source Chinese Hamster Ovary cell line, CHO-derived human /PCSK2 protein Gly110-Asn638 with a C-terminal 6-His tag Accession # P16519 N-terminal Sequence Gly110 Analysis Predicted Molecular 59 kDa Mass

SPECIFICATIONS SDS-PAGE 66-82 kDa, reducing conditions Activity Measured by its ability to cleave the fluorogenic peptide substrate pERTKR-AMC (Catalog # ES013). The specific activity is >180 pmol/min/μg, as measured under the described conditions. Endotoxin Level <1.0 EU per 1 μg of the protein by the LAL method. Purity >90%, by SDS-PAGE under reducing conditions and visualized by silver stain.

Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and CaCl2. See Certificate of Analysis for details.

Activity Assay Protocol

Materials Assay Buffer: 50 mM NaOAc, 100 mM NaCl, 1 mM CaCl2, 0.5% (w/v) Brij-35, pH 5.0 Recombinant Human Proprotein Convertase 2/PCSK2 (rhPCSK2) (Catalog # 6018-SE) Substrate: L-PYROGlu-Arg-Thr-Lys-Arg-AMC (Catalog # ES013), 4 mM stock in deionized water F16 Black Maxisorp Plate (Nunc, Catalog # 475515 Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

Assay 1. Dilute rhPCSK2 to 0.4 µg/mL in Assay Buffer. 2. Dilute Substrate to 500 µM in Assay Buffer. 3. Start the reaction by mixing equal volumes of 0.4 µg/mL rhPCSK2 and 500 µM Substrate together and incubate at 37 °C for 30 minutes. For a Substrate Blank, mix Assay Buffer and Substrate together and incubate at 37 °C for 30 minutes. 4. Into a plate, load 100 µL of the reaction and Substrate Blank (in triplicate). 5. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in endpoint mode. 6. Calculate specific activity: Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU) Specific Activity (pmol/min/µg) = Incubation time (min) x amount of (µg) *Adjusted for Substrate Blank **Derived using calibration standard 7-Amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A-9891).

Final Assay Per Well: Conditions rhPCSK2: 0.02 µg Substrate: 250 µM

PREPARATION AND STORAGE Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. Stability & Storage Use a manual defrost freezer and avoid repeated freeze-thaw cycles. 6 months from date of receipt, -70 °C as supplied. 3 months, -70 °C under sterile conditions after opening.

BACKGROUND Proprotein Convertase 2 (PCSK2) is a -like serine peptidase that processes proteins into biologically active products in the secretory pathway. Like other / family members, PCSK2 cleaves its substrates primarily after sites of paired basic amino acid residues (1). Autoactivation of PCSK2 occurs in a post- Golgi compartment of the secretory system (2). Interaction with the secretory protein 7B2 is required for proper activation of PCSK2 (3). The N-terminal domain of 7B2 stabilizes active PCSK2, and a C-terminal fragment of 7B2 can inhibit PCSK2 activity (4). rhPCSK2 was coexpressed with 7B2 to facilitate activation of the enzyme. PCSK2 is a major proteolytic processing enzyme of the regulated secretory pathway of the neuroendocrine system, where it generates a number of and neuropeptides (5). Products of PCSK2 processing include enkephalins, , somatostatin, , and LHRH.

References: 1. Zhou, A. et al. (1999) J. Biol. Chem. 274:20745. 2. Lamango, N.S. et al. (1999) Arch. Biochem. Biophys. 362:275. 3. Zhu, X. and I. Lindberg (1995) J. Cell Biol. 129:1641. 4. Lamango, N. et al. (1996) Arch. Biochem. Biophys. 330:238. 5. Rouillé, Y. et al. (1995) Front. Neuroendocrinol. 16:322.

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