METAP2/Methionine aminopeptidase 2 Protein, Human (sf9, His)

METAP2/Methionine aminopeptidase 2 protein removes N-terminal methionine from nascent proteins. It preferentially cleaves when the second residue is small and uncharged. METAP2 has higher catalytic activity for Met-Val peptides than METAP1, suggesting its role in processing Met-Val and Met-Thr sequences in vivo. It also protects EIF2S1 from phosphorylation by inhibitory kinases, regulating protein synthesis. METAP2/Methionine aminopeptidase 2 Protein, Human (sf9, His) is the recombinant human-derived METAP2/Methionine aminopeptidase 2 protein, expressed by sf9 insect cells , with N-6*His labeled tag.

METAP2/Methionine aminopeptidase 2 protein functions by cotranslationally removing the N-terminal methionine from nascent proteins. This N-terminal methionine cleavage occurs when the second residue in the primary sequence is small and uncharged, such as Met-Ala-, Cys, Gly, Pro, Ser, Thr, or Val. The catalytic activity of human METAP2 towards Met-Val peptides is significantly higher than that of METAP1, suggesting its responsibility for processing proteins containing N-terminal Met-Val and Met-Thr sequences in vivo. In addition, METAP2 protects eukaryotic initiation factor EIF2S1 from translation-inhibiting phosphorylation by inhibitory kinases like EIF2AK2/PKR and EIF2AK1/HCR, thus playing a critical role in regulating protein synthesis.

Measure by its ability to remove methionine from a fluorogenic peptide substrate H-Met-Gly-Pro-AMC. The resulting GP-AMC is cleaved by Recombinant Human DPPIV/CD26 that incubate at room temperature in kinetic mode for 5 minutes. The specific activity is >15 pmol/min/µg.

Materials
Assay buffer: 50 mM HEPES, 0.1 mM CoCl₂, 100 mM NaCl, pH 7.5
METAP2/Methionine aminopeptidase 2 Protein, Human (sf9, His) (HY-P70364)
CD26/Dipeptidyl Peptidase 4 Protein, Human (HEK293, His) (HY-P70017)
Substrate: Met-Gly-Pro-AMC (HY-P4344)
Standard: 7-Amino-4-Methyl-Courmarin (HY-D0027)

Procedure
1. Standard curve: Dilute standard to 0, 6.25, 12.5, 25, 50, 100 μmol/L with assay buffer, and add 100 μL of each into the black zymography well. The excitation and emission wavelengths were 380 nm and 460 nm, respectively, and read in kinetic mode for 5 min. The measured RFU was used as the horizontal coordinate, and the amount of standard substance was used as the vertical coordinate to make a standard curve and obtain the standard curve formula.
2. Dilute Human METAP2 to 50 μg/mL with assay buffer.
3. Dilute the substrate to 500 μM with 2 μg/mL Human CD26 in assay buffer.
4. Experimental group: 50 μg/mL of Human METAP2L 50 μL was loaded into the plate and 50 μL of substrate/Human CD26 mixture was added to start the reaction.
Control group: 50 μL substrate/Human CD26 mixture + 50 μL buffer.
5. Incubate for 10 minutes at room temperature.
6. Read in kinetic mode for 5 minutes at excitation and emission wavelengths of 380 nm and 460 nm, respectively.
7. Calculate the specific activity:

Human

Sf9 insect cells

N-6*His

P50579-1 (A2-Y478)

10988  [NCBI]

Full Length of Isoform-1 Mature Protein

AGVEEVAASGSHLNGDLDPDDREEGAASTAEEAAKKKRRKKKKSKGPSAAGEQEPDKESGASVDEVARQLERSALEDKERDEDDEDGDGDGDGATGKKKKKKKKKRGPKVQTDPPSVPICDLYPNGVFPKGQECEYPPTQDGRTAAWRTTSEEKKALDQASEEIWNDFREAAEAHRQVRKYVMSWIKPGMTMIEICEKLEDCSRKLIKENGLNAGLAFPTGCSLNNCAAHYTPNAGDTTVLQYDDICKIDFGTHISGRIIDCAFTVTFNPKYDTLLKAVKDATNTGIKCAGIDVRLCDVGEAIQEVMESYEVEIDGKTYQVKPIRNLNGHSIGQYRIHAGKTVPIVKGGEATRMEEGEVYAIETFGSTGKGVVHDDMECSHYMKNFDVGHVPIRLPRTKHLLNVINENFGTLAFCRRWLDRLGESKYLMALKNLCDLGIVDPYPPLCDIKGSYTAQFEHTILLRPTCKEVVSRGDDY

Approximately 58-70 kDa due to the glycosylation.

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

Solution.

Supplied as a 0.22 μm filtered solution of 20 mM Tris, 500 mM NaCl, 10% glycerol, pH 8.0.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<1 EU/μg, determined by LAL method.

N/A.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.