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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
OTKA
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2.

001-es BibID:BIBFORM084744
Első szerző:Szigeti Márton (környezetmérnök)
Cím:Rapid N -glycan release from glycoproteins using immobilized PNGase F microcolumns / Szigeti Marton, Bondar Judit, Gjerde Douglas, Keresztessy Zsolt, Szekrenyes Akos, Guttman Andras
Dátum:2016
ISSN:1570-0232
Megjegyzések:N-glycosylation profiling of glycoprotein biotherapeutics is an essential step in each phase of product development in the biopharmaceutical industry. For example, during clone selection, hundreds of clones should be analyzed quickly from limited amounts of samples. On the other hand, identification of disease related glycosylation alterations can serve as early indicators (glycobiomarkers) for various pathological conditions in the biomedical field. Therefore, there is a growing demand for rapid and easy to automate sample preparation methods for N-glycosylation analysis. In this paper, we report on the design and implementation of immobilized recombinant glutathione-S-transferase (GST) tagged PNGase F enzyme microcolumns for rapid and efficient removal of N-linked carbohydrates from glycoproteins. Digestion speed and efficiency were compared to conventional in-solution based protocols. The use of PNGase F functionalized microcolumns resulted in efficient N-glycan removal in 10 min from all major N-linked glycoprotein types of: (i) neutral (IgG), (ii) highly sialylated (fetuin), and (iii) high mannose (ribonuclease B) carbohydrate containing glycoprotein standards. The approach can be readily applied to automated sample preparation systems, such as liquid handling robots.
Tárgyszavak:Orvostudományok Klinikai orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
N-glycosylation
Enzyme immobilization
PNGase F
Capillary electrophoresis
Megjelenés:Journal Of Chromatography B-Analytical Technologies In The Biomedical And Life Sciences. - 1032 (2016), p. 139-143. -
További szerzők:Bondar Judit Gjerde, Douglas Keresztessy Zsolt Szekrényes Ákos (1983-) (vegyészmérnök) Guttman András (1954-) (vegyészmérnök)
Pályázati támogatás:NKFIH-K116263
Egyéb
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