Recombinant Escherichia coli Metalloprotease/StcE His, CF Summary
| Additional Information |
His-tag |
| Details of Functionality |
Measured by its ability to cleave Recombinant CD45 Protein at specific O-glycan sites. One μg of Recombinant E. coli StcE will cleave >60% of Recombinant CD45 Protein with labeled O-glycan
(Catalog #
1430-CD), as measured under the described conditions. |
| Source |
E. coli-derived e. coli StcE protein
Ala36-Lys898 with a N-terminal Met and 6-His tag |
| Accession # |
|
| N-terminal Sequence |
Met & Arg415 |
| Protein/Peptide Type |
Recombinant Enzymes |
| Purity |
>85%, 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 |
|
| Theoretical MW |
97 kDa & 54 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 |
86-95 & 53-58 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 Tris and NaCl. |
| Purity |
>85%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining. |
| Assay Procedure |
- Digestion Buffer: 50 mM Tris, 150 mM NaCl, pH 7.5
- Labeling Buffer: 50 mM HEPES, 10 mM MnCl2, pH 7.0
- Recombinant E. coli StcE His-Tag (rE. StcE) (Catalog # 11406-MP)
- Recombinant Human CD45 (rhCD45) (Catalog #
1430-CD)
- Recombinant Human ST3GAL2 (rhST3GAL2) (Catalog #
7275-GT)
- Recombinant C. perfringens Neuraminidase (Catalog #
5080-NM)
- CMP-Cy3-Sialic Acid (Cy3-SA) (Catalog #
ES402)
- 12% SDS-PAGE gel
- Gel loading dye
- Gel Imager with Cy3 fluorescent dye detection capability
- Dilute rhCD45 to 200 µg/mL and rE. StcE to 100 µg/mL with Digestion Buffer.
- Combine 10 µL of 200 µg/mL rhCD45 and 10 µL of 100 µg/mL rE StcE. For a control, combine 10 µL of Digestion Buffer and 10 µL of 200 µg/mL rhCD45.
- Incubate at 37 °C for 2 hours.
- Heat tubes for 2 minutes at 95 °C.
- Create a labeling mixture containing 10 µg/mL rhST3GAL2, 2.5 µg/mL of Neuraminidase, and 10 µM of Cy3-SA with Labeling Buffer.
- Add 20 µL of labeling mixture to each reaction and control.
- Incubate at 37 °C for 1 hour.
- Add Loading dye to each tube.
- Load half the volume of each reaction and control onto a 12% 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 | ) ] x 100 | | Total Control Intensity |
Per Reaction: - rE. StcE: 1 µg
- rhCD45: 2 µg
- rhST3GAL2: 0.2 µg
- rCp Neuraminidase: 0.05 µg
- CMP-Cy3-Sialic Acid: 0.2 nmol
|
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Escherichia coli Metalloprotease/StcE His, CF
Background
Recombinant
E. coli StcE (secreted protease of C1-esterase inhibitor) is a zinc-dependent, monomeric mucinase from Enterohaemorrhagic Escherichia coli (EHEC). EHEC is a major foodborne pathogen that must penetrate a thick mucus layer for effective infection. StcE has three distinct globular domains with non-catalytic globular domains at the N- and C-terminus and the catalytic metalloprotease domain in the middle. This open arrangement is thought to allow for additional regulatory functions and flexibility to recognize large O-glycosylated substrates (1, 2). StcE is secreted via the type II general secretory pathway during infection as a major virulence factor due to its protease-meditated activity towards heavily glycosylated proteins with high levels of O-linkages that play a host defensive role such as mucin 7 and glycoprotein 340, in addition to C1-esterase inhibitor, a regulator of inflammation pathways (2). StcE has also been implicated in cleavage of glycoproteins found on the surface of various cells such as intestinal epithelial cells and neutrophils, where it has been shown to lead to depression of neutrophil migration during infection (3, 4). StcE has been proposed as a treatment for cystic fibrosis as it is has been shown to loosen mucus in affected patients (1, 4). In addition, StcE has been proposed as a useful tool for the analysis of mucin-domain glycoproteins known to be important in several human diseases including cancer (5-8). StcE has a broad specificity from simple to complex O-glycans, with specificity for a discrete peptide- and glycan-based motif that allows its utilization in glycoproteomic mapping of mucin glycosites or enrichment of mucins (5-8). The activity of recombinant StcE is demonstrated in an electrophoretic gel mobility shift assay using enzymatically fluorophore labeled mucin-like glycoproteins.
- Yu, A. et al. (2012). Structure. 20:707.
- Grys, TE. et al. (2005). Infection and Immunity. 73:1295.
- Furniss, RCD. et al. (2018). J Biol. Chem. 293:17188.
- Hews, CL et al. (2017). Cell. Microbiol. 19:e12717.
- Malaker, SA. et al. (2019). Proc. Natl. Acad. Sci. USA. 116:7278.
- Taleb, V. et al. (2022). Nat. Commun. 13:4324.
- Shon, DJ. et al. (2020). Proc. Natl. Acad. Sci. USA. 117:21299.
- Pluvinage, B. et al. (2021). Proc. Natl. Acad. Sci. USA. 118:e2019220118.
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