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Purication of Peptide-N – (N- acetyl- β- D glucosaminyl) Asparagines amidase F for Deglycosylation Studies of Glycoproteins

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Author(s): Vishranti A.A | Pallavi T. K

Journal: Advanced Biotech
ISSN 0973-0109

Volume: 13;
Issue: 02;
Start page: 15;
Date: 2013;
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Keywords: PNGase F | Chromatography | Enterokinase | Monoclonal antibody | Deglycosylation

ABSTRACT
Glycosylation is the most wide spread form of post-translation odication of proteins. Glycosidases are the enzymes used to get nformation about the carbohydrate groups attached to glycoproteins and glycopeptides. 4 The study was aimed to Purify Peptide-N – (N- acetyl- β- D glucosaminyl) asparagines amidase F (PNGase F) produced by recombinant E.coli fermentation as MAP-PNGase F fusion protein. Soluble MAP-PNGase F fusion protein was isolated by cell lysis and centrifugation. MAP-PNGase F was separated from other Host Cell Proteins, Host cell DNAand other major impurities using anion exchange chromatography (Diethylaminoethyl sepharose). PNGase F was separated from MAP (Methionine amino peptidase) by Enterokinase digestion. Further Purication was achieved by anion exchange chromatography (Diethylaminoethyl sepharose) and cation exchange chromatography (Sulfopropyl sepharose). This removed trace impurities and concentrated the product. The nal purity of PNGase F achieved by the process was greater than 95%. Puried PNGase F successfully deglycosylated Puried monoclonal antibody by non-denaturing method and Enterokinase by denaturing method respectively
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