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AuthorLauber, Jenniferdc.contributor.author
Date of accession2016-03-15T10:40:56Zdc.date.accessioned
Available in OPARU since2016-03-15T10:40:56Zdc.date.available
Year of creation2015dc.date.created
AbstractRecombinant protein therapeutics have revolutionised the treatment of many diseases and are produced using well-established expression systems based on bacteria, yeast, insect and mammalian cells. Approximately 40 % of therapeutic proteins are glycoproteins and therefore the post-translational attachment of sugar residues is required. However, the use of bacteria for expression of eukaryotic proteins is limited by the lack of post-translational modification pathways, including those for glycosylation. The development of an Escherichia coli-based model system for production of human glycoproteins could potentially lead to increased yields, as well as significant decreases in processing time and costs. Aim of this work was the expression of functional human-derived glycosyltransferase UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 2 (GalNAcT2) in a recombinant E. coli strain. The gene encoding amino acids 52-571 of GalNAcT2 was codon-optimised and subsequently inserted into a pET-23 derived expression vector encoding a polyhistidine-tag which was translationally fused to the N-terminus of the glycosyltransferase (HisDapGalNAcT2). The glycosyltransferase HisDapGalNAcT2 was produced in SHuffle® T7 host cells using a recently published expression system including the pre-expression of the sulfhydryl oxidase Erv1p and protein disulfide isomerase PDI. Soluble HisDapGalNAcT2 was purified using Ni-NTA-chromatography and subsequently analysed by SEC-MALS and CD spectroscopy. The enzymatic activity of purified HisDapGalNAcT2 was monitored using a colorimetric assay by using a model acceptor peptide or, alternatively, the granulocyte-colony stimulating growth factor (G-CSF). The successful in vitro glycosylations were assessed by MALDI-TOF-MS analysis and ESI-MS analysis. The results indicated the presence of N-acetylgalactosamine residues at several putative glycosylation sites of the model acceptor peptide and at one position of the acceptor peptide G-CSF.dc.description.abstract
Languagededc.language.iso
PublisherUniversität Ulmdc.publisher
LicenseStandarddc.rights
Link to license texthttps://oparu.uni-ulm.de/xmlui/license_v3dc.rights.uri
KeywordEnzymatic activity glycosyltransferasedc.subject
KeywordErv1p/PDI pre-expressiondc.subject
KeywordGlycosyltransferase GalNAcT2dc.subject
KeywordIn vitro glycosylationdc.subject
KeywordProtein expressiondc.subject
KeywordSecondary structure glycosyltransferasedc.subject
Dewey Decimal GroupDDC 570 / Life sciencesdc.subject.ddc
LCSHG-CSF (Colony-stimulating factor)dc.subject.lcsh
LCSHGlycosyltransferase genesdc.subject.lcsh
TitleExpression einer funktionellen rekombinanten Glykosyltransferase in Escherichia colidc.title
Resource typeDissertationdc.type
DOIhttp://dx.doi.org/10.18725/OPARU-3348dc.identifier.doi
PPN843602120dc.identifier.ppn
URNhttp://nbn-resolving.de/urn:nbn:de:bsz:289-vts-97985dc.identifier.urn
GNDDNS-Glycotransferasedc.subject.gnd
GNDEscherichia colidc.subject.gnd
FacultyFakultät für Naturwissenschaftenuulm.affiliationGeneral
Date of activation2015-12-04T09:07:51Zuulm.freischaltungVTS
Peer reviewneinuulm.peerReview
Shelfmark print versionW: W-H 14.482uulm.shelfmark
DCMI TypeTextuulm.typeDCMI
VTS-ID9798uulm.vtsID
CategoryPublikationenuulm.category
University Bibliographyjauulm.unibibliographie


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