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Gewählte Doctoral Thesis:

Kristof Zarschler (2009): Development of a model organism for bacterial surface layer protein O-glycosylation.
Doctoral Thesis - Studienabteilung, BOKU-Universität für Bodenkultur, pp 201. UB BOKU obvsg FullText

Data Source: ZID Abstracts
Abstract:
Posttranslational glycosylation is the most prevalent and most diverse modification of proteins. Surface (S-) layer glycoproteins are among the best-studied prokaryotic glycoproteins. Most S-layer (glyco)proteins have the unique feature to self-assemble into two-dimensional crystalline arrays on the supporting cell envelope. Paenibacillus alvei CCM 2051T possesses a glycosylated S-layer with known glycan structure. Its O-glycan is a heteropolymer, which is linked to specific tyrosine residues of the S-layer protein SpaA. To gain insights into the mechanisms governing S-layer glycan biosynthesis, a ~24.3-kb gene cluster responsible for glycan biosynthesis (slg gene cluster) has been identified, sequenced, and the genes have been assigned by protein database comparison. To investigate in detail the function of individual enzymes encoded by the slg gene cluster, a reliable protocol for introduction of plasmid-DNA into P. alvei CCM 2051T by electroporation, a shuttle vector for heterologous gene expression, and an effective gene knockout system for insertional inactivation of desired target genes were developed. All ORFs of the gene cluster except those encoding nucleotide sugar biosynthesis enzymes and the ABC transporter integral transmembrane protein were disrupted by insertion of the mobile group II intron, and glycosylated S-layer proteins produced in mutant backgrounds were analyzed by mass spectrometry. These results, combined with the observed similarity of the respective proteins to database entries, allowed an almost complete assignment of their biological function. Additionally, the S-layer structural gene spaA has been sequenced and used for surface display of chimeric S-layer glycoproteins. These data clearly demonstrate the feasibility of manipulating the glyco-dimension of an S-layer based construction kit for life- and non-life science application.


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