Endo F1

Endo F1, Endoglycosidase F1, endo-beta-N-acetylglucosaminidase F

Endo F1 cleaves Asparagine-linked high mannose and some hybrid oligosaccharides. Core fucosylation reduces the activity by 50 fold. Endoglycosidase F1 will hydrolyze sulfate containing high-mannose chains. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact.

Additional Endo F Products
Endoglycosidase F2 releases biantennary and high mannose glycans (at a 40X reduced rate)
Endoglycosidase F3 will release triantennarry and fucosylated biantennary N-glycans
The Endo F Multi-Kit includes 20 µLs of each of the Endo F enzymes and their buffers.

Source recombinant Elizabethkingia miricola (was Chryseobacterium meningosepticum) in E. Coli

EC 3.2.1.96

Endo F1 Specifictity Cleaves all asparagine-linked high mannose and some hybrid oligosaccharides

Contents
60 µl aliquot of enzyme (1 U) in 20 mM Tris-HCl, pH 7.5

Included with 20 µL and 60 µL pack sizes:
5x Reaction Buffer – 250 mM sodium phosphate, pH 5.5

Specific Activity >16 U/mg
Activity >17 U/ml

Molecular weight 32,000 daltons

Suggested usage
1. Add up to 200 µg of glycoprotein to an Eppendorf tube.
2. Add deionized water to a total of 33 µL.
3. Add 10 µL 5X Reaction Buffer, 5.5.
4. Add 2.5 µL of Denaturation Solution. Heat at 90ºC for 10 minutes.
5. Cool to room temperature and add 2.5 µL Triton X-100 solution.
6. Add 2 µL of Endoglycosidase F1. Incubate 1 hour or more at 37ºC.
7. Monitor cleavage by SDS-PAGE.

For digestion of native proteins, add water to a total volume of 38 µL and omit steps 4 and 5. Increase incubation time appropriately.

Specific Activity
Defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of denatured Ribonuclease B (RNase B) in 1 minute at 37˚C, pH 5.5. Cleavage is monitored by SDS-PAGE (cleaved RNase B migrates faster).

Storage Store enzyme at 4˚C.

Stability Stable at least 24 months when stored properly. Several days exposure to ambient tempertures will not reduce activity.

Purity Endoglycosidase F1 is tested for contaminating protease as follows; 10 μg of denatured BSA is incubated for 24 hours at 37oC with 2 μL of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation.

The production host strain has been extensively tested and does not produce any detectable glycosidases.

Endo F2 References:

Maley P., R. B. Trimble, A. L. Tarentino and T. H. Plummer Jr. Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Anal Biochem 180:195-204 (1989).

Plummer, T. H. Jr, A. W. Phelan and A. L. Tarentino. Porcine fibrinogen glycopeptides: substrates for detecting endo-N-acetylglucosaminidases F2 and F3. Anal Biochem 235:98-101 (1996).

Reddy A., B. G. Grimwood, T. H. Plummer Jr and A. L. Tarentino. High- level expression of the Endo-beta-N- acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity. Glycobiology 8:633-636 (1998).

Tarentino, A. L., C. M. Gomez and T. H. Plummer Jr. Deglycosylation of Asparagine-Linked Glycans by Peptide:N-Glycosidase F. Biochemistry 24:4665-4671 (1985).

Tarentino A. L., G. Quinones, W. P. Schrader, L. M. Changchien and T. H. Plummer Jr. Multiple endoglycosidase (Endo) F activities expressed by Flavobacterium meningosepticum. Endo F1: molecular cloning, primary sequence, and structural relationship to Endo H. J Biol Chem 267:3868-3872 (1992).

Tarentino A. L., G. Quinones, L. M. Changchien, and T. H. Plummer Jr. Multiple endoglycosidase F activities expressed by Flavobacterium meningosepticum endoglycosidases F2 and F3: Molecular cloning, primary sequence, and enzyme expression. J Biol Chem 268(13):9702-9708 (1993).

Tarentino A. L. and T. H. Plummer Jr. Substrate specificity of Flavobacterium meningosepticum: Endo F2 and endo F3: purity is the name of the game. Glycobiology 4:771-773 (1994).

Tarentino, A. L. and T. H. Plummer Jr. Enzymatic deglycosylation of asparagine- linked glycans: purification, properties and specificity of oligosaccharide- cleaving enzymes from Flavobacterium meningosepticum. Methods in Enzymology 230:44-57 (1994).

Tarentino A. L., G. Quinones and T. H. Plummer Jr. Overexpression and purification of non-glycosylated recombinant endo-beta-N- acetylglucosaminidase F3. Glycobiology 5:599-601 (1995).

Trimble, R. B. and A. L. Tarentino. Identification of Distinct Endoglycosidase (Endo) Activities in Flavobacterium meningosepticum: Endo F1, Endo F2 and Endo F3. J. Biol Chem 266:1646-1651 (1991).