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3. 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).
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Endo F2 cleaves N-linked (asparagine-linked) biantennary oligosaccharides from glycoproteins. It also will cleave high mannose glycans 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.
Endoglycosidase F2 is less sensitive to protein conformation than PNGase F and is therefore more suitable for deglycosylation of native proteins. However, for optimal results, denaturation of the glycoprotein is recommended.
Recombinant gene from Elizabethkingia miricola in E. Coli
Kit includes enzyme plus reaction buffer. Sufficient for up to 60 reactions.
Source: Recombinant Elizabethkingia miricola in E. Coli
Alternative Names: Endo F2, Endoglycosidase F2, endo-β-N-acetylglucosaminidase F2
Cleaves all asparagine-linked biantennary oligosaccharides and high mannose (though at a 40X reduced rate) N-glycans from peptides and proteins
60 µL aliquot of enzyme (0.3 U) in 10 mM sodium acetate 25mM NaCl, pH 4.5
5x Reaction Buffer - 250 mM sodium acetate, pH 4.5
Specific Activity: >20 U/mg
Activity: >5 U/mL
Molecular weight: 32,000 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
3. Add 2.0 µL of Endo F2. 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 denatured porcine fibrinogen in 1 minute at 37°C, pH 5.5. Cleavage is monitored by SDS-PAGE (cleaved finbrinogen 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 F2 is tested for contaminating protease as follows; 10 µg of denatured BSA is incubated for 24 hours at 37°C 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.