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@article{1165396,
author = {Krenkova, Jana and Szekrenyes, Akos and Keresztessy, Zsolt and Foret, František and Guttman, Andras},
article_location = {AMSTERDAM},
article_number = {Dec},
doi = {http://dx.doi.org/10.1016/j.chroma.2013.10.087},
keywords = {Enzyme microreactor; Oriented immobilization; Monolith; PNGase F; Deglycosylation},
language = {eng},
issn = {0021-9673},
journal = {Journal of Chromatography A},
title = {Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis},
volume = {1322},
year = {2013}
}
TY - JOUR
ID - 1165396
AU - Krenkova, Jana - Szekrenyes, Akos - Keresztessy, Zsolt - Foret, František - Guttman, Andras
PY - 2013
TI - Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis
JF - Journal of Chromatography A
VL - 1322
IS - Dec
SP - 54-61
EP - 54-61
PB - Elsevier
SN - 00219673
KW - Enzyme microreactor
KW - Oriented immobilization
KW - Monolith
KW - PNGase F
KW - Deglycosylation
N2 - 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 succinimidy1-6-(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. (C) 2013 Elsevier B.V All rights reserved.
ER -
KRENKOVA, Jana, Akos SZEKRENYES, Zsolt KERESZTESSY, František FORET a Andras GUTTMAN. Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis. \textit{Journal of Chromatography A}. AMSTERDAM: Elsevier, 2013, roč.~1322, Dec, s.~54-61. ISSN~0021-9673. Dostupné z: https://dx.doi.org/10.1016/j.chroma.2013.10.087.
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