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Purification of endo-N-acetyl-β-d-glucosaminidase H by substrate-affinity chromatography

✍ Scribed by Urs F. Greber; Branko Kozulić; Klaus Mosbach

Publisher
Elsevier Science
Year
1989
Tongue
English
Weight
815 KB
Volume
189
Category
Article
ISSN
0008-6215

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✦ Synopsis

Endo-N-acetyl-P-D-glucosaminidase H (Endo H) was purified to homogeneity (3000-fold) from a culture filtrate of Streptomyces plicatus. The key step was substrate-affinity chromatography, which afforded a lOOO-fold purification and yielded a protease-and exoglycosidase-free preparation of Endo H. Proteins from the crude sample were applied to the substrate-affinity column, consisting of yeastinvertase glycopeptides bound to Sepharose-immobilized concanavalin A. After washing off the unbound proteins, Endo H was quantitatively eluted by methyl cu-D-mannopyranoside.

Various conditions were tested to achieve an optimal binding of Endo H to this substrate-affinity gel. After substrate-affinity chromatography, Endo H was separated from the coeluted glycopeptide substrate and some protein impurities by gel filtration and hydrophobic chromatography.

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