Recombinant Coliphage K5 Lyase His-tag Protein, CF
Recombinant Coliphage K5 Lyase His-tag Protein, CF Summary
Product Specifications
Ala2-Lys632 with a N-terminal Met and 6-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
11384-K5
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and DTT. |
| Shipping | The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Assay Buffer: 50 mM Sodium Acetate, pH 4.5
- Recombinant Coliphage K5 Lyase His-tag (rC. K5 Lyase) (Catalog # 11384-K5)
- Cy5-labeled Heparan Sulfate Proteoglycan (Cy5-HSPG) (Catalog # GL402)
- 15% SDS-PAGE
- Gel loading dye
- Gel Imager with Cy5 fluorescent dye detection capability
- Dilute rC. K5 Lyase to 100 µg/mL with Assay Buffer.
- Dilute Cy5-HSPG to 0.2 µM with Assay Buffer.
- For reaction, combine 10 µL of rC. K5 Lyase and 10 µL of Cy5-HSPG. For a control, combine 10 µL of Assay Buffer and 10 µL of Cy5-HSPG.
- Incubate reaction and control at 37 °C for 60 minutes.
- Add gel loading dye to each tube.
- Load half the volume of each reaction and control onto a 15% SDS-PAGE gel. Let samples migrate at least 80% down the gel before stopping.
- Acquire gel image and determine percent cleavage.
| % Cleavage = [ 1 - ( | Intensity of uncleaved Cy5-HSPG | ) ] x 100 |
| Total Control Intensity |
- rC. K5 Lyase: 1 µg
- Cy5-HSPG: 2 pmol
Scientific Data
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Recombinant Coliphage K5 Lyase His-tag Protein (Catalog # 11384-K5) has specificity for the repeating disaccharide [-4)-GlcA-beta (1,4)-GlcNAc-alpha (1-] of heparosan, which is similar to heparin and heparan sulfate.
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2 μg/lane of Recombinant Coliphage K5 Lyase His-tag Protein (Catalog # 11384-K5) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 54-60 kDa under reducing conditions.
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Using Cy5-labeled Heparan Sulfate Proteoglycan (Cy5-HSPG) (GL402) as a substrate Recombinant Coliphage K5 Lyase Protein, CF (Catalog # 11384-K5) cleaves intact heparan sulfate proteoglycans into heparan sulfate (HS) oligos. SDS-PAGE gel was imaged using the red fluorescent channel.
Reconstitution Calculator
Background: Coliphage K5 Lyase
Recombinant Coliphage K5 Lyase His-tag (K5 Lyase) is a viral tail spike protein from the K5A bacteriophage that infects the K5 strain of Escherichia coli (1). The overall structure of mature K5 Lyase comprises a trimer of three identical monomers with the monomers aligned and intertwined at the carboxyl‑terminal domain. Each monomer consists of a small N‑terminal alpha ‑helical domain, a central single-stranded, right-handed beta -helix domain, and a C‑terminal beta ‑prism/triple-stranded beta -helix domain (2). K5 Lyase is specifically active on heparosan, a glycosaminoglycan with the repeating disaccharide [-4)-GlcA-beta (1,4)-GlcNAc-alpha (1-], that comprises the polysaccharide capsule of E. coli that must be degraded by the virus to permit penetration of the capsule (3). The strict specificity of K5 Lyase means it does not recognize hyaluronan, an isomer of the K5 polysaccharide, and is only capable of cleaving alpha 1‑4 (not beta 1‑4) hexosaminidic linkages (4). K5 Lyase is of particular interest in the field of heparin and heparan sulfate glycobiology because both glycans contain the same core linear disaccharide structure of heparosan with varying sulfation. As heparin and heparan sulfate play important roles in blood coagulation, cell proliferation, and development (5,6), K5 lyase can be used as an enzymatic tool to cleave and modulate these important glycans. The activity of K5 Lyase is demonstrated in an electrophoretic gel mobility shift assay using Cy5‑Labeled Heparan Sulfate Proteoglycan.
- Hanfling, P. et al. (1998) J. Bact. 178:4747.
- Thompson, J.E. et al. (2010) J. Bio. Chem. 285:23963.
- O' Leary, T.R. et al. (2013) Glycobiology. 23:132.
- Murphy, K.J. et al. (2004) J. Bio. Chem. 279:27239.
- Linhardt, R.J. et al. (2003) J. Med. Chem. 46:2551.
- Chen, J. et al. (2007) Chem. & Biol. 9:986.
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