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2.4.99.18: dolichyl-diphosphooligosaccharide-protein glycotransferase

This is an abbreviated version!
For detailed information about dolichyl-diphosphooligosaccharide-protein glycotransferase, go to the full flat file.

Word Map on EC 2.4.99.18

Reaction

dolichyl diphosphooligosaccharide
+
[protein]-L-asparagine
=
dolichyl diphosphate
 +
a glycoprotein with the oligosaccharide chain attached by N-beta-D-glycosyl linkage to a protein L-asparagine

Synonyms

AglB, AglB-L, AlgB, asparagine N-glycosyltransferase, DGL1, dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST2, dolichyl-diphosphooligosaccharide-protein glycosyltransferase, dolichyldiphosphooligosaccharide-protein oligosaccharyltransferase, EC 2.4.1.119, glycosyltransferase, dolichyldiphosphooligosaccharide-protein, glycosyltransferase, dolichylpyrophosphodiacetylchitobiose-protein, L.m.STT3, Lm STT3, oligomannosyltransferase, oligosaccharide transferase, oligosaccharyl transferase, oligosaccharyl transferase 16 kDa subunit, oligosaccharyl transferase subunit epsilon, oligosaccharyl transferase subunit OST2, oligosaccharyltransferase, oligosaccharyltransferase complex, oligosaccharyltransferase STT3, oligosaccharyltransferase, dolichyldiphosphoryloligosaccharide-protein, OST, OST-A, OST-B, OST-I, OST-II, OST-III, OST1, OST2, OST3, Ost3p, OST4, OST5, Ost6p, OSTC(I), OSTC(II), OSTC(III), OTase, PglB, PglB oligosaccharyltransferase, PglB1, PglL, PilO, STT3, STT3 protein, STT3-1, STT3-2, STT3-3, STT3-4, Swp1p, TbSTT3A, TbSTT3B, TbSTT3C, Wbp1p, yeast oligosaccharyl transferase

ECTree

     2 Transferases
         2.4 Glycosyltransferases
             2.4.99 Transferring other glycosyl groups
                2.4.99.18 dolichyl-diphosphooligosaccharide-protein glycotransferase

Purification

Purification on EC 2.4.99.18 - dolichyl-diphosphooligosaccharide-protein glycotransferase

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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
affinity purification of a tagged Stt3 protein (component of the oligosaccharyltransferase complex)
C-terminal domain of Stt3p, wild-type and mutant D518E
cells centrifuged, resuspended, and washed in 20 mM phosphate buffer, pH 7.2, with 0.3 M Na Cl, cells disrupted and centrifuged, membranes separated by ultracentrifugation of supernatant, resuspended in buffer with 2% Elugent and 25 mM imidazole buffer, proteins solubilized by tumbling, centrifuged, supernatant removed, Elugenat reduced to 1% or 0.5% n-dodecyl-beta-D-maltopyranoside, loaded onto Ni-nitrilotriacetic acid agarose column, elution with buffer containing 250 mM imidazole
-
dissolution in denaturing buffer (6 M guanidine hydrochloride, 500 mM Na Cl, 25 mM imidazole, 20 mM phosphate buffer, pH 7.4), cenrtrifugation, supernatant loaded onto nickel-NTA column for affinity chromatography with 20 mM phosphate buffer, pH 6.5, SDS-PAGE
Ni-NTA resin column chromatography
-
partial
purification of an affinity-tagged version of the enzyme complex from a membrane protein fraction
-
recombinant N-terminally His6-tagged wild-type and selenomethionine-labeled AglB from Escherichia coli strain BL21 by nickel affinity chromatography and gel filtration, followed by anion exchange chromatography
sonication, centrifugation, supernatant absorbed to glutathione-Sepharose 4B resin, elution, concentration, reductive methylation, gel filtration with Superdex75 column, followed by anion exchange chromatography with a Resource Q column