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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 and Andras GUTTMAN. Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis. \textit{Journal of Chromatography A}. AMSTERDAM: Elsevier, 2013, vol.~1322, Dec, p.~54-61. ISSN~0021-9673. Available from: https://dx.doi.org/10.1016/j.chroma.2013.10.087.
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