Recombinant Coliphage K5 Lyase His-tag Protein, CF

Catalog #: 11384-K5 Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
11384-K5-020

Recombinant Coliphage K5 Lyase His-tag Protein Enzyme Activity Diagram.

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All Coliphage K5 Lyase Proteins and Enzymes

Product Details

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Coliphage K5 Lyase His-tag Protein, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to cleave the repeating disaccharide of Heparan Sulfate.

Recombinant K5 Lyase will cleave >80% Cy5-labeled HSPG (Catalog # GL402), as measured under the described conditions.

Source

E. coli-derived Coliphage K5 Lyase protein
Ala2-Lys632 with a N-terminal Met and 6-His tag

Accession #

YP_654147.1

N-terminal Sequence
Analysis

Met

Predicted Molecular Mass

68 kDa

SDS-PAGE

54-60 kDa, under reducing conditions.

Product Datasheets

11384-K5

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials

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

Per Reaction:

rC. K5 Lyase: 1 µg
Cy5-HSPG: 2 pmol

Scientific Data

Enzyme Activity

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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.

SDS-PAGE

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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.

Enzyme Activity

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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.

References