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001-es BibID:BIBFORM050938
Első szerző:Krenkova, Jana
Cím:Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis / Jana Krenkova, Akos Szekrenyes, Zsolt Keresztessy, Frantisek Foret, Andras Guttman
Dátum:2013
ISSN:0021-9673
Megjegyzések: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.
Tárgyszavak:Természettudományok Kémiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
Enzyme microreactor
Oriented immobilization
Monolith
PNGase F
Deglycosylation
Megjelenés:Journal of Chromatography A. - 1322 (2013), p. 54-61. -
További szerzők:Szekrényes Ákos (1983-) (vegyészmérnök) Keresztessy Zsolt Foret, František Guttman András (1954-) (vegyészmérnök)
Pályázati támogatás:K-81839
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