Endo F3, Endoglycosidase F3, endo-beta-N-acetylglucosaminidase F
Endo F3 cleaves free or Asparagine-linked triantennary or alpha-(1-6) fucosylated biantennary oligosaccharides,as well as triamnnosyl chitobiose core structures. Nonfucosylated biantennary glycans will also be cleaved, but at a 40x reduced rate. 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. Alpha 1-3 fucosylation will inhibit enzymatc activity. There is no activity on oligomannose and hybrid molecules.
Additional Endo F Products
Endoglycosidase F1 releases bianatennary and some hybrid glycans
Endoglycosidase F2 will release high mannose and biantennary N-glycans
The Endo F Multi-Kit includes 20 µLs of each of the Endo F enzymes and their buffers.
Source recombinant Elizabethkingia meningosepticum (was Chryseobacterium meningosepticum)
Cleaves all asparagine-linked triantennary or alpha-(1-6)-fucosylated biantennary oligosaccharides, as well as triamannosyl chitobiose core structures. Alpha 1-3 fucosylation will inhibit enzymatic activity. Nonfucosylated biantennary glycans will also be cleaved, but at a 40x reduced rate. (Note:
The recombinant version is not glycosylated, which may result in properties differing from the native protein.)
60 µl aliquot of enzyme (0.33 U) in 20 mM Tris-HCl, pH 7.5
5x Reaction Buffer – 250 mM sodium acetate, pH 4.5
Specific Activity >25 U/mg
Activity >5 U/ml
Molecular weight 30,000 daltons
1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 38 µl final volume with de-ionized water.
2. Add 10 µl 5x Reaction Buffer 4.5
4. Add 2.0 µl of Endo F3. Incubate 1 hour at 37˚C.
Defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of porcine fibrinogen in 1 minute at 37˚C, pH 4.5. Cleavage is monitored by SDS-PAGE (cleaved fibrinogen migrates faster).
Storage Store enzyme at 4˚C.
Stability Stable at least 12 months when stored properly. Several days exposure to ambient tempertures will not reduce activity.
Purity Endoglycosidase F3 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 F3 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).