Ludger recombinant PNGase F enzyme structure

    Recombinant PNGase F


    E-rPNG01

    PNGase F is suitable for release of all types (high-mannose, hybrid and complex) N-linked glycans from glycoproteins and glycopeptides. PNGase F will not remove oligosaccharides containing α(1-3) linked core fucose commonly found on plant glycoproteins.


      View product documentation: Specsheet / CofA / MSDS

    • Part Number
    • E-rPNG01
    • Amount of Enzyme
    • 0.3 U / 60 µL








    Kit includes enzyme plus reaction buffers.
    Sufficient for up to 60 reactions.






















    Product Specifications:


    Recombinant PNGase F is isolated from a E. coli strain containing a clone of the Elizabethkingia miricola gene. There is no detectable difference in activity or specific activity of the recombinant enzyme from the native enzyme (E-PNG01).

    PNGase F cleaves asparagine-linked (N-linked) oligosaccharides from glycoproteins. PNGase F deaminates asparagine to aspartic acid, leaving the oligosaccharides intact. Denaturation increases the rate of cleavage up to 100x. Most native proteins can still be completely N-deglycosylated but incubation time must be increased. PNGase F will remain active under incubation conditions for at least 72 hours. PNGase F will not remove oligosaccharides containing α(1,3)-linked core fucose commonly found on plant glycoproteins; for this purpose, use peptide N-glycosidase A.

    PNGase F Source: recombinant PNGase F gene from Elizabethkingia miricola in E. coli

    EC: 3.5.1.52

    Alternative Names: PNGase F, Peptide N Glycosidase F, N-Glycosidase, N-Glycanase

    Contents:
    Ludger recombinant PNGase F - Kit contents
    60 µL aliquot of recombinant PNGase F (0.3 U) in 20 mM Tris-HCl, pH 7.5
    5x PNGase F Reaction Buffer 7.5 for PNGase F - 250 mM sodium phosphate, pH 7.5
    PNGase F Denaturation Solution - 2% SDS, 1 M Beta-mercaptoethanol
    PNGase F Triton X-100 - 15% solution

    Specific Activity: >25 U/mg

    Activity: 5 U/mL

    Molecular weight: 36,000 daltons

    pH range for PNGaseF: 6-10, optimum 7.5

    (Protocol) PNGaseF suggested usage:
    1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 35 µL final volume with de-ionized water.
    2. Add 10 µL 5x PNGase F Reaction Buffer 7.5 and 2.5 µL of PNGase F Denaturation Solution. Heat at 100°C for 5 minutes.
    3. Cool. Add 2.5 µL of PNGaseF Triton X-100 and mix.
    4. Add 2.0 µL of PNGaseF to the reaction. Incubate 3 hours at 37°C.

    Specificity: PNGase F cleaves asparagine-linked (N-linked) oligosaccharides from glycoproteins. PNGase F deaminates asparagine to aspartic acid, leaving the oligosaccharides intact. Denaturation increases the rate of cleavage up to 100x. Most native proteins can still be completely N-deglycosylated but incubation time must be increased. PNGase F will remain active under incubation conditions for at least 72 hours. PNGase F will not remove oligosaccharides containing α(1,3)-linked core fucose commonly found on plant glycoproteins; for this purpose, use peptide N-glycosidase A.

    Specific Activity: Defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of denatured RNase B in 1 minute at 37°C, pH 7.5. Cleavage is monitored by SDS-PAGE (cleaved RNase B migrates faster).

    Storage: Store enzyme at 4°C.





Companion Products:

Process controls


Glycan clean-up - removal of proteins and enzymes from glycan mixtures following endoglycosidase treatment

  • LC-PBM-96
  • LudgerClean Protein Binding Membrane (96 samples)

Labelling and derivatisation of released glycans - View our Glycan Labelling Summary Table

  • LT-KAB-A2
  • LT-KAB-VP24
  • LT-KAB-VP96
  • LT-KPROC-24
  • LT-KPROC-96
  • LT-KPROC-VP24
  • LT-KAA-A2
  • LT-KAA-VP24
  • LT-PERMET-96
  • LT-PERMET-VP96
  • LudgerTag 2-AB Glycan Labelling Kit, sodium cyanoborohydride (24 samples)
  • LudgerTag 2-AB Glycan Labelling Kit, picoline borane (24 samples)
  • LudgerTag 2-AB Glycan Labelling Kit, picoline borane (96 samples)
  • LudgerTag Procainamide Glycan Labelling Kit, sodium cyanoborohydride (24 samples)
  • LudgerTag Procainamide Glycan Labelling Kit, sodium cyanoborohydride (96 samples)
  • LudgerTag Procainamide Glycan Labelling Kit, picoline borane (24 samples)
  • LudgerTag 2-AA Glycan Labelling Kit, sodium cyanoborohydride (24 samples)
  • LudgerTag 2-AA Glycan Labelling Kit, picoline borane (24 samples)
  • LudgerTag Permethylation Kit (96 samples)
  • LudgerTag Permethylation Kit, without methyl iodide (96 samples)

Alternative enzymes

  • E-EF01
  • E-EF02
  • E-EF03
  • E-EH02
  • E-G001
  • KE-DG01
  • KE-DGMX
  • Endoglycosidase F1
  • Endoglycosidase F2
  • Endoglycosidase F3
  • Endoglycosidase H
  • O-glycosidase
  • Enzymatic CarboRelease Kit - contains enzymes necessary for removing N-linked and O-linked oligosaccharides
  • Enzymatic DeGlycoMx Kit - contains enzyme mix for removing N-linked and O-linked oligosaccharides






References:

1. Bayer, E.A., F. De Meester, T. Kulik and M. Wilchek. Preparation of deglycosylated egg white avidin. Appl Biochem Biotech 53: 1-9 (1995)

2. Elder, J.H. and S. Alexander. endo-b-N-Acetylglucosaminidase F: endoglycosidase from Flavobacterium meningosepticum that cleaves both high-mannose and complex glycoproteins. Proc Natl Acad Sci USA 79: 4540-4544 (1982)

3. 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)

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

5. Trimble R.B. and A.L. Tarentino. Identification of distinct endoglycosidase (endo) activities in Flavobacterium meningosepticum: endo F1 , endo F2 and endo F3. Endo F1 and endo H hydrolyze only high mannose and hybrid glycans. J Biol Chem 266: 1646-1 651 (1991)

6. Taga, E. M., A. Waheed and R. L. Van Etten. Structural and chemical characterization of a homogeneous peptide-N-glycosidase from almond. Biochemistry 23: 815-22 (1984)

7. Tarentino AL, Trimble RB, Plummer TH. Enzymatic approaches for studying the structure, synthesis, and processing of glycoproteins. Methods in Cell Biology: 32: 111-39 (1989)

8. Anthony L. , Tarentino and Thomas 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)

9. Tarentino AL, Plummer TH. Oligosaccharide accessibility to peptide:N-glycosidase as promoted by protein-unfolding reagents. The Journal of Biological Chemistry. 257 (18): 10776-80. (1982)