Recombinant S. agalactiae Hyaluronan Lyase Protein, CF

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

Recombinant S. agalactiae Hyaluronan Lyase Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the native substrate Hyaluronan (Catalog # GLR004). The specific activity is >75,000 pmol/min/µg, as measured under the described conditions.
• Source: E. coli-derived s. agalactiae Hyaluronan Lyase protein
  Ser259-Ile1072, with an N-terminal Met and 6-His tag
  Accession # NP_688206
• Accession #: NP_688206
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• 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: 93 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: 86 kDa, 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 under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 500 mM Sodium Acetate, 50 mM CaCl₂, pH 6.5
  • Recombinant Streptococcus agalactiae Hyaluronan Lyase (rHyluronan Lyase) (Catalog # 5150-GH)
  • Substrate: Hyaluronan (Catalog # GLR004)
  • 96 Well UV Transparent Plate (Costar, Catalog # 3635)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute Substrate to 5.0 mg/mL in Assay Buffer.
  2. Dilute rHyaluronan Lyase to 2.0 µg/mL in Assay Buffer.
  3. Load into a plate 50 µL of 2.0 µg/mL rHyaluronan Lyase, and start the reaction by adding 50 µL of 5.0 mg/mL Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 5.0 mg/mL Substrate.
  4. Read plate at 232 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/M) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 3800 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD Per Well:
  • rHyluronan Lyase: 0.1 µg
  • Substrate: 2.5 mg/mL

Notes

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

Alternate Names for Recombinant S. agalactiae Hyaluronan Lyase Protein, CF

• Hyaluronan Lyase

Background

The hyaluronan lyase from Streptococci agalactiae is highly specific towards hyaluronan (1). However, a thousand fold lower activity towards unsulfated chondroitin sulfate has also been observed (2). The enzyme activity is Ca²⁺ dependent (3). During digestion the enzyme moves processively along the hyaluronan chains continuously releasing disaccharide units as it travels (1). The recombinant enzyme can be used to digest hyaluronan and study chondroitin sulfate structure (2). Unlike hyaluronidases, hyaluronan lyase digestion of the substrate creates a double bond at the non-reducing terminus of the product; therefore, the digestion progress can be followed by UV spectrometry and the enzyme can be used for hyaluronan quantification. The recombinant enzyme can also be used for drug screening because the enzyme is a major pathogenic factor for Streptococci agalactiae that causes serious, often fatal, neonatal infections (4). The expressed protein corresponds to the mature form of the native protein (3).

1. Lin, B. et al. (1994) J. Biol. Chem. 269:30113.
2. Baker, J. et al. (1997) Biochem. J. 327:65.
3. Akhtar, M. et al. (2006) J. Biol. Chem. 281:28336.
4. Hynes, W.L. and Walton, S.L. (2000) FEMS Microbiol. Lett. 183:201.

Publications for Hyaluronan Lyase (5150-GH)(1)

Publication using 5150-GH

• Wu Z, Person A, Anderson M, Burroughs B, Tatge T, Khatri K, Zou Y, Wang L, Geders T, Zaia J, Sackstein R Imaging specific cellular glycan structures using glycosyltransferases via click chemistry. Glycobiology, 2018-02-01;0(0):. 2018-02-01 [PMID: 29186441] (Click Chemistry, Human, Mouse)

Bioinformatics

• Uniprot:
  • NP_688206()