Title of article :
Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis
Author/Authors :
Krenkova، نويسنده , , Jana and Szekrenyes، نويسنده , , Akos and Keresztessy، نويسنده , , Zsolt and Foret، نويسنده , , Frantisek and Guttman، نويسنده , , Andras، نويسنده ,
Issue Information :
روزنامه با شماره پیاپی سال 2013
Abstract :
In this paper, we report on a novel oriented peptide-N-glycosidase F (PNGase F) immobilization approach onto methacrylate based monolithic support for rapid, reproducible and efficient release of the N-linked carbohydrate moieties from glycoproteins. The glutathione-S-transferase-fusion PNGase F (PNGase F-GST) was expressed in Escherichia coli using regular vector technology. The monolithic pore surface was functionalized with glutathione via a succinimidyl-6-(iodoacetyl-amino)-hexanoate linker and the specific affinity of GST toward glutathione was utilized for the oriented coupling. This novel immobilization procedure was compared with reductive amination technique commonly used for non-oriented enzyme immobilization via primary amine functionalities. Both coupling approaches were compared using enzymatic treatment of several glycoproteins, such as ribonuclease B, fetuin and immunoglobulin G followed by MALDI/MS and CE-LIF analysis of the released glycans. Orientedly immobilized PNGase F via GST-glutathione coupling showed significantly higher activity, remained stable for several months, and allowed rapid release of various types of glycans (high-mannose, core fucosylated, sialylated, etc.) from glycoproteins. Complete protein deglycosylation was obtained as fast as in several seconds when using flow-through immobilized microreactors.
Keywords :
Enzyme microreactor , monolith , PNGase F , deglycosylation , Oriented Immobilization
Journal title :
Journal of Chromatography A
Journal title :
Journal of Chromatography A