Recombinant Human PSMA/FOLH1/NAALADase I Avi Fc Protein, CF

Biotinylated Recombinant Human PSMA/FOLH1/ NAALADase I Avi-tag Fc Chimera Protein (Catalog # AVI11535) binds Human PSMA/FOLH1/ NAALADase I Antibody (MAB4234) with an ED50 of 0.0750‑1.20 μg/mL.

2 μg/lane of Biotinylated Recombinant Human PSMA/FOLH1/NAALADase I Avi-tag Fc Chimera Protein (Catalog # AVI11535) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 112-125 kDa, under reducing conditions.

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

Recombinant Human PSMA/FOLH1/NAALADase I Avi Fc Protein, CF Summary

• Additional Information: Fc Chimera
• Details of Functionality: Measured by its ability to hydrolyze the substrate N-acetyl-L-Asp-L-Glu into N-acetyl-L-Asp and L-Glu. The L-Glu product is measured by fluorescence after its derivatization by ortho-phthaldialdehyde. The specific activity is >325 pmol/min/μg, as measured under the described conditions. Measured by its binding ability in a functional ELISA. Biotinylated Recombinant Human PSMA/FOLH1/NAALADase I Avi-tag Fc Chimera (Catalog # AVI11535) binds Human PSMA/FOLH1/NAALADase I Antibody (Catalog # MAB4234) with an ED₅₀ of 0.0750-1.20 μg/mL.
• Source: Chinese Hamster Ovary cell line, CHO-derived human PSMA/FOLH1/NAALADase I protein
  

  Avi-tag	MD	Human IgG1 (Pro100-Lys330)	IEGR	Human NAALADase-1 (Lys44-Ala750) Accession # Q04609.1
  N-terminus				C-terminus

• Accession #: Q04609.1
• N-terminal Sequence: Gly
• Structure / Form:

  Biotinylated via Avi-tag

• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Binding Activity2
• Theoretical MW: 108 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: 112-125 kDa, under reducing conditions.

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in MES and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM HEPES, 100 mM NaCl, pH 7.5
  • o-PA Buffer: 0.2 M NaOH containing 0.1% beta -Mercaptoethanol (v/v)
  • Recombinant Human PSMA/FOLH1/NAALADase-1/N- Avitag/Fc (rhPSMA) (Catalog # AVI11535)
  • Substrate: Ac-Asp-Glu, 10 mM stock in 40 mM NaOH
  • o-phthaldialdehyde (o-PA), 50 mg/mL (373 mM) stock in DMSO
  • Black 96-well Plate
  • Plate Reader with Fluorescence Read Capability
  1. Dilute rhPSMA to 0.4 µg/mL in Assay Buffer.
  2. Dilute Substrate to 40 µM in Assay Buffer.
  3. Combine 125 µL of 0.4 µg/mL rhPSMA and 125 µL of 40 µM Substrate. For a control, inactivate 125 µL of 0.4 µg/mL rhPSMA by heating it at 95 °C for 5 minutes, then add 125 µL of 40 µM Substrate.
  4. Incubate reactions and controls at 37 °C for 60 minutes.
  5. Stop the reaction by heating reactions and controls at 95 °C for 5 minutes, then cool to room temperature.
  6. Prepare a 15 mM o-PA solution in o-PA Buffer.
  7. Add 250 µL of 15 mM o-PA solution to each reaction and control. Vortex and incubate at room temperature for 10 minutes.
  8. Load 200 µL of reactions and controls to plate.
  9. Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively, in endpoint mode.
  10. Calculate specific activity:

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

  *Adjusted for Control
  **Derived using calibration standard L-Glutamic Acid
  Per Well:
  • rhPSMA: 0.02 µg
  • Substrate: 10 µM
  • o-PA: 7.5 mM

Notes

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

Alternate Names for Recombinant Human PSMA/FOLH1/NAALADase I Avi Fc Protein, CF

• Cell growth-inhibiting gene 27 protein
• cell growth-inhibiting protein 27
• EC 3.4.17.21
• FGCP
• folate hydrolase (prostate-specific membrane antigen) 1
• Folate hydrolase 1
• FOLH1
• FOLHNAALADase I
• Folylpoly-gamma-glutamate carboxypeptidase
• GCP2
• GCPII
• GCPIImGCP
• glutamate carboxylase II
• glutamate carboxypeptidase 2
• Glutamate carboxypeptidase II
• Membrane glutamate carboxypeptidase
• mopsm
• NAALAD1
• NAALAD1N-acetylated-alpha-linked acidic dipeptidase I
• NAALADase I
• NAALAdase
• N-acetylated alpha-linked acidic dipeptidase 1
• prostate specific membrane antigen variant F
• Prostate-specific membrane antigen
• PSMA
• PSMAFGCP
• PSMPteroylpoly-gamma-glutamate carboxypeptidase

Background

Prostate-specific membrane antigen (PSMA), a tumor marker in prostate cancer encoded by the FOLH1 gene, is a type II transmembrane zinc metallopeptidase that is most highly expressed in the nervous system, prostate, kidney, and small intestine (1,2). PMSA has a short cytosolic N-terminal domain, a single membrane-spanning segment, and an extracellular region that is composed of a protease domain, apical domain, and C-terminal domain (3). The extracellular domains all contribute to substrate recognition. The protein forms an active homodimer reliant on interactions between amino-acid side chains and glycosylation (3,4). PSMA is also known as glutamate carboxypeptidase II (GCPII), folate hydrolase 1, and N-acetylated-alpha-linked acidic dipeptidase-1 (NAALADase1). PSMA activity plays a role in tumor angiogenesis making it not only a tumor marker, but a therapeutic target in cancers including prostate cancer (5). In the brain, PSMA hydrolyzes the neurotransmitter N-acetyl-Asp-Glu (NAAG) to produce glutamate, another neurotransmitter. Inhibition of brain PSMA activity is considered to be a promising approach for the treatment of neurological disorders associated with glutamate excitotoxicity such as stroke, schizophrenia, Alzheimer's, and amyotrophic lateral sclerosis (5,6,7). Intestinal PSMA hydrolyzes folylpoly-gamma -glutamates, facilitating the uptake of folate (8). Upregulation of PSMA is present in inflammatory bowel disease, Crohn's disease, and ulcerative colitis where pharmacological inhibition has shown amelioration of clinical symptoms pertaining to these diseases in mice (5).

1. Silver, D.A. et al. (1997) Clin. Cancer Res. 3:81.
2. Carter, R.E. et al. (1996) Pro. Natl. Acad. Sci. USA. 93:749.
3. Mesters, J.R. et al. (2006) EMBO J. 25:1375.
4. Shulke, N. et al. (2003) Proc. Natl. Acad. Sci. USA 100:12590.
5. Vornov, J.J. et al. (2020) Neurochem. Res. 45:1256.
6. Jackson, P.F. and Slusher, B.S. (2001) Curr. Med. Chem. 8:949.
7. Neale, J. J and T. Yamamoto. (2020) Prog. Neurobiol. 184:101722.
8. Heston, W.D. (1997) Urology 49:104. 

Bioinformatics

• Uniprot:
  • Q04609.1()