03-237 | Furin (Human) Recombinant

03-237
600 units  
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      Overview

      Replacement Information

      Key Spec Table

      Uni Prot Number
      P09958
      Description
      Catalogue Number03-237
      Trade Name
      • Chemicon
      DescriptionFurin (Human) Recombinant
      OverviewFormat
      Recombinant secreted human furin protein, catalytic domain, C-terminally tagged with V5 and 6xHis in PBS with 50% glycerol.

      Method of Expression and Purification
      Stably expressed in MDCK cells. Self-cleaved, secreted protein is purified from media by Nickel-chelate chromatography.
      Background InformationFurin is a ubiquitous subtilisin-like proprotein convertase that is the major processing enzyme of the secretory pathway, localized in the trans-Golgi network. The structure of furin is a type I membrane protein consisting of a heterogeneous ~10 kDa amino-terminal proregion, a highly conserved ~55 kDa subtilisin-like catalytic domain, and carboxyl-terminal domain that is heterogeneous in length and sequence. As a calcium-dependent, serine protease, it processes matrix metalloproteinases stromelysin-3 (MMP-11) and MT1-MMP, as well as pro-hormones, neuropeptide precursors, some viral proteins, and bacterial toxins that are transported by the constitutive secretory pathway. Cleavage of target proteins occurs at the carboxyl-terminus of the furin consensus sequence, RX(K/R)R. It has been shown that the acidic peptide sequence, C(771)PSDSEEDEG(780), localizes furin to the trans-Golgi-network. Phosphorylation of serine residues within this region modulates intracellular routing of furin protein. An additional signaling domain includes the tetrapeptide sequence, Y(759)KGL(762), which directs internalization from the cell surface.
      References
      Product Information
      Components
      • 2 vials of Cat. # 03-183 (15 µL each vial, 600 Units Total)

      • Unit Definition:
        One unit is defined as the amount of furin that will cleave 1.0 pmol AMC from the substrate pGlu-Arg-Thr-Lys-Arg-AMC per min at 30°C, pH 7.5.
      ActivityUnit Definition
      One unit is defined as the amount of furin that will cleave 1.0 pmol AMC from the substrate pGlu-Arg-Thr-Lys-Arg-AMC per min at 30°C, pH 7.5.

      Unit Assay Conditions
      Furin activity was measured in a 96-well plate in 50µL of 50 mM HEPES, pH 7.5, containing 1 mM CaCl2, 0.005% Brij35 and 20% Glycerol. Pyr-Arg-Thr-Lys-Arg-methyl-coumaryl-7-amide (25 µM) was used as a fluorescent substrate. The steady-state rate of substrate hydrolysis was monitored continuously (λex =360 nm and λem =465 nm) at 37 °C using a fluorescent spectrophotometer (Wallac Victor 3, Perkin Elmer).
      Applications
      Application NotesPurified Furin protein gel image.
      10 µL of eluted furin solution was loaded onto 10-20% Bis-Tris gel and stained with SafeBlue.
      (See Figure 1)

      Furin activity titration.
      A 2-fold serial dilution of Furin was applied with 25 µM fluorescent substrate (EMD Millipore, Cat#344935) at 37 °C for 60 minutes. A linear enzyme activity was plotted using GraphPad Prism. (See Figure 2)

      Furin activity time course.
      15U of Furin was monitored continuously (λex =360 nm and λem =465 nm) with 25µM fluorescent substrate (EMD Millipore, Cat#344935) at 37 °C. The steady-state rate of substrate hydrolysis was approaching at ~60 minutes. (See Figure 3)

      Furin inhibitor IC 50 value determination.
      15U of Furin was applied with two furin specific inhibitors, inhibitor I (EMD Millipore, Cat# 34490) and inhibitor II (EMD Millipore, Cat# 344931) using 25µM fluorescent substrate (EMD Millipore, Cat#344935) at 37 °C for 60 minutes. IC50 value was determined by non-linear fit using GraphPad Prism. (See Figure 4)
      Biological Information
      Purity> 90%, by SDS-PAGE under reducing conditions and visualized by safeblue.
      SpecificityThe minimal cleavage site is Arg-X-X-Arg. However, the enzyme prefers the site Arg-X-(Lys/Arg)-Arg.
      Gene Symbol
      • FURIN
      Purification MethodNickel-chelate chromatography
      UniProt Number
      UniProt SummaryFUNCTION: Furin is likely to represent the ubiquitous endoprotease activity within constitutive secretory pathways and capable of cleavage at the RX(K/R)R consensus motif.
      CATALYTIC ACTIVITY: Release of mature proteins from their proproteins by cleavage of -Arg-Xaa-Yaa-Arg-|-Zaa- bonds, where Xaa can be any amino acid and Yaa is Arg or Lys. Releases albumin, complement component C3 and vWF from their respective precursors.
      COFACTOR: Calcium
      ENZYME REGULATION: Could be inhibited by the not secondly cleaved propeptide.
      SUBUNIT: Interacts with FLNA. Binds to PACS1 which mediates TGN localization and connection to clathrin adapters.
      SUBCELLULAR LOCATION: Golgi apparatus › trans-Golgi network membrane; Single-pass type I membrane protein. Cell membrane; Single-pass type I membrane protein. Note: Shuttles between the trans-Golgi network and the cell surface. Propeptide cleavage is a prerequisite for exit of furin molecules out of the endoplasmic reticulum (ER). A second cleavage within the propeptide occurs in the trans Golgi network (TGN), followed by the release of the propeptide and the activation of furin.
      TISSUE SPECIFICITY: Seems to be expressed ubiquitously.
      DOMAIN: Contains a cytoplasmic domain responsible for its TGN localization and recycling from the cell surface.
      PTM: The inhibition peptide, which plays the role of an intramolecular chaperone, is autocatalytically removed in the endoplasmic reticulum (ER) and remains non-covalently bound to furin as a potent autoinhibitor. Following transport to the trans Golgi, a second cleavage within the inhibition propeptide results in propeptide dissociation and furin activation.
      Phosphorylation is required for TGN localization of the endoprotease. In vivo, exists as di-, mono- and non-phosphorylated forms.
      SIMILARITY: Belongs to the peptidase S8 family. Furin subfamily. Contains 1 homo B/P domain.
      Molecular Weight~60 kDa
      Physicochemical Information
      ActivityUnit Definition
      One unit is defined as the amount of furin that will cleave 1.0 pmol AMC from the substrate pGlu-Arg-Thr-Lys-Arg-AMC per min at 30°C, pH 7.5.

      Unit Assay Conditions
      Furin activity was measured in a 96-well plate in 50µL of 50 mM HEPES, pH 7.5, containing 1 mM CaCl2, 0.005% Brij35 and 20% Glycerol. Pyr-Arg-Thr-Lys-Arg-methyl-coumaryl-7-amide (25 µM) was used as a fluorescent substrate. The steady-state rate of substrate hydrolysis was monitored continuously (λex =360 nm and λem =465 nm) at 37 °C using a fluorescent spectrophotometer (Wallac Victor 3, Perkin Elmer).
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Usage Statement
      • Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
      Storage and Shipping Information
      Storage ConditionsStore at -80°C. Avoid freeze/thaw cycles.
      Packaging Information
      Material Size600 units
      Transport Information
      Supplemental Information
      Specifications

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