Recombinant Human Methionine Aminopeptidase 1 Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Methionine Aminopeptidase 1 Protein, CF Summary

• Details of Functionality: Measure by its ability to remove methionine from a fluorogenic peptide substrate H-Met-Gly-Pro-AMC (Catalog # ES017). The resulting GP-AMC is cleaved by Recombinant Human DPPIV/CD26 (Catalog # 9168-SE). The specific activity is >200 pmol/min/µg, as measured under the described conditions. S
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Methionine Aminopeptidase 1/METAP1 protein
  His52-Phe386 & Lys53-Phe386, with a C-terminal 10-His tag
• Accession #: P53582
• N-terminal Sequence: His52 & Lys53
• Protein/Peptide Type: Recombinant Enzymes
• Gene: METAP1
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 38 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 38 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • Activation Buffer: 50 mM HEPES, 0.1 mM CoCl₂ , 0.1 M NaCl, pH 7.5
  • Assay Buffer: 25 mM Tris, pH 8.0
  • Recombinant Human Methionine Aminopeptidase 1/METAP1 (rhMETAP1) (Catalog # 3537-ZN)
  • Recombinant Human DPPIV/CD26 (rhCD26) (Catalog # 9168-SE)
  • Substrate Met-Gly-Pro-AMC (Catalog # ES017)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhMETAP1 to 4 µg/mL in Activation Buffer.
  2. Dilute Substrate to 200 µM in Activation Buffer.
  3. Combine equal volumes of 4 µg/mL rhMETAP1 and 200 µM Substrate. Include a Substrate Blank containing Activation Buffer in place of rhMETAP1.
  4. Incubate reactions for 5 minutes at room temperature.
  5. Stop reactions by heating at 95-100 °C for 5 minutes.
  6. Cool reactions on ice for 3 minutes and centrifuge briefly.
  7. Dilute rhCD26 to 2 ng/µL in Assay Buffer.
  8. Load into a black well plate 50 µL of each incubated mixture and add 50 µL 2 ng/µL rhCD26 to each.
  9. Incubate at room temperature for 10 minutes.
  10. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in endpoint mode.
  11. Calculate specific activity using the following formula:

  Specific Activity (pmol/min/µg) = (Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU)) / (Incubation time (min) x amount of enzyme (µg))

  *Adjusted for Substrate Blank

  **Derived using calibration standard 7-Amino-4-Methyl-Courmarin (AMC) (Sigma, Catalog # A-9891).

  Per Well:
  • rhMETAP1: 0.10 µg
  • rhCD26: 0.10 µg
  • Substrate: 50 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Methionine Aminopeptidase 1 Protein, CF

• DKFZp781C0419
• EC 3.4.11.18
• KIAA0094MAP 1
• MAP1
• MAP1A
• MetAP 1
• MetAP1
• MetAP1A
• Methionine Aminopeptidase 1
• methionyl aminopeptidase 1
• Peptidase M 1

Background

The human METAP1 gene encodes Methionine Aminopeptidase 1, a member of the M24 family of metalloproteases. METAPs catalyze the removal of the initiator methionine residue from nascent peptides (1) and are essential for cell growth (2). Inhibition of METAPs provides a novel strategy in developing anti-cancer drugs (3). The purified rhMETAP1 consists of amino acid residues 52/53 to 386. A previous report showed that the N-terminal 89 amino acid region was not essential for catalytic activity (4).

1. Lowther, W.T. and B.W. Matthews (2000) Biochim. Biophys. Acta. 1477:157.
2. Li, X. and Y.H. Chang (1995) Proc. Natl. Acad. Sci. USA 92:12357.
3. Liu, S. et al. (1998) Science 282:1324.
4. Addlagatta, A. et al. (2005) Biochemistry 44:14741.

Bioinformatics

• Gene Symbol: METAP1
• Uniprot:
  • P53582()