Recombinant Human Sialate O-acetylesterase/SIAE His-tag, CF

Recombinant Human SIAE His-tag Protein (Catalog # 11497-SI) catalyzes the removal of O-acetyl ester groups from position 9 of Sialic Acid.

Recombinant Human SIAE His-tag Protein (Catalog # 11497-SI) is measured by its ability to hydrolyze p-nitrophenylacetate.

2 μg/lane of Recombinant Human SIAE His-tag Protein (Catalog # 11497-SI) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 63-69 & 88-97 kDa, under reducing conditions.

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

Recombinant Human Sialate O-acetylesterase/SIAE His-tag, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze p-nitrophenylacetate. The specific activity is >25000 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human SIAE protein
  Ile24-Lys523, with a C-terminal 6-His tag
• Accession #: Q9HAT2
• N-terminal Sequence: Ile24
• 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:
  • Enzyme Activity
• Theoretical MW: 57 kD.
  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: 63-69 & 88-97 kD, 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 Tris and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, pH 8.0
  • Recombinant Human SIAE His tag (rhSIAE) (Catalog # 11497-SI)
  • Substrate: 4-Nitrophenyl Acetate, 100 mM stock in acetone
  • Clear 96 Well Plate
  • Plate Reader with Absorbance Read Capability
  1. Dilute rhSIAE to 1.2 μg/mL in Assay Buffer.
  2. Dilute Substrate to 8 mM in Assay Buffer.
  3. Load into a plate 50 μL of 1.2 μg/mL rhSIAE, and start the reaction by adding 50 μL of 8 mM Substrate. For Substrate Blank, load 50 μL of Assay Buffer and 50 μL of 8 mM Substrate.
  4. Read plate at 410 nm in kinetic mode for 5 minutes.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  *Adjusted for Substrate Blank
  **Using the extinction coefficient 18500 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD
  Per Well:
  • rhSIAE: 0.06 μg
  • Substrate: 4 mM

Notes

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

Alternate Names for Recombinant Human Sialate O-acetylesterase/SIAE His-tag, CF

• AIS6
• Bakers yeast
• Baker's yeast
• Bakers' yeast
• Brewers yeast
• Brewer's yeast
• Brewers' yeast
• CSEC
• CSE-C
• EC 3.1.1.53
• LSE
• MGC87009
• S cerevisiae
• S. cerevisiae
• S.cerevisiae
• SIAE
• Sialate O-Acetylesterase
• YSG2

Background

Recombinant human Sialate O-acetylesterase (SIAE) is a glycosylated lysosomal protein that has two transcript isoforms: the cytosolic sialic acid esterase (Cse) that lacks 35 amino acids at the N-terminus and the lysosomal/membrane-associated sialic acid esterase (Lse) (1, 2). The Lse form is the predominantly expressed form responsible for the acetyl esterase activity on NeuAc in most tissues (2) and represents the recombinant product form. SIAE contains a signal sequence, a variable N-terminus, and a core protein structure that is unique within the SGNH hydrolase family (2) and contains a shallow active site pocket that accommodates binding of various complex substrates with acetylated sialic acid. SIAE Lse form catalyzes the removal of O-acetyl ester groups from position 9 of Sialic acid and is highly expressed in the testis, colon, and prostrate (3). SIAE deficiency has been shown to impact B cell development, signaling, and immunological tolerance through modulation of Siglec binding (4). Many SIAE variants in humans that result in defective activity or improper secretion have been identified in patients with autoimmune diseases including Crohn's disease, Sjogren's syndrome, lupus, rheumatoid arthritis, and type I diabetes (5). In addition, SIAE modulation of Siglec binding and sialic acid levels may play a key role in the immune evasion pathway associated with cancer (6). SIAE has also been implicated to play an important role in immune modulation during pregnancy (7). 

1. Higa, H.H. et. al. (1987) Biochem. Biophys. Res. Commun. 144:1099.
2. Orizio, F. et. al. (2015) Glycobiology 9:992.
3. Zhu, H. et. al. (2004) J. Biomed. Biotechnol. 3:130.
4. Cariappa, A. et. al. (2009) J. Exp. Med. 206:125.
5. Surolia, I. et. al. (2010) Nature 466:243.
6. Grabenstein, S. et. al. (2021) Glycobiology 31:1279.
7. Erickson, J. et. al. (2022) Nature 606:769.

Bioinformatics

• Uniprot:
  • Q9HAT2()