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Martin-Luther-Universität Halle-Wittenberg

5 ARTICLES PUBLISHED IN JoVE

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Bioengineering

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines
Paul R. Wratil 1,2, Rüdiger Horstkorte 3
1Max von Pettenkofer-Institut & Genzentrum, Virologie, Nationales Referenzzentrum für Retroviren, Medizinische Fakultät, LMU München, 2Institut für Laboratoriumsmedizin, klinische Chemie und Pathobiochemie, Charité - Universitätsmedizin Berlin, 3Institut für Physiologische Chemie, Martin-Luther-Universität Halle-Wittenberg

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

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Bioengineering

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence
Tobias Baumann 1, Franz-Josef Schmitt 2, Almut Pelzer 1, Vivian Jeanette Spiering 3, Georg Johannes Freiherr von Sass 1, Thomas Friedrich 2, Nediljko Budisa 1
1Institute of Chemistry L 1, Department of Biocatalysis, Technical University of Berlin, 2Institute of Chemistry PC 14, Department of Bioenergetics, Technical University of Berlin, 3Institute of Chemistry TC 7, Department of Physical Chemistry/Molecular Material Sciences, Technical University of Berlin

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed "gold" fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

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Bioengineering

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids
Jessica H. Nickling *1, Tobias Baumann *1, Franz-Josef Schmitt 2, Maike Bartholomae 3, Oscar P. Kuipers 3, Thomas Friedrich 2, Nediljko Budisa 1
1Department of Biocatalysis, Institute of Chemistry, Technische Universität Berlin, 2Department of Bioenergetics, Institute of Chemistry, Technische Universität Berlin, 3Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Department of Molecular Genetics, University of Groningen

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

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Immunology and Infection

Analyzing the Permeability of the Blood-Brain Barrier by Microbial Traversal through Microvascular Endothelial Cells
Veronika Weber 1, Kaya Bork 1, Rüdiger Horstkorte 1, Heidi Olzscha 1
1Institute of Physiological Chemistry, Martin Luther University Halle-Wittenberg

The human blood-brain barrier selectively prevents penetration of hydrophilic molecules and pathogens into the brain. Several pathologies, including meningitis and postoperative delirium, are associated with an increased permeability of the blood-brain barrier. Here, we describe an endothelial cell culture model to test the barrier permeability by microbial traversal.

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Bioengineering

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability
Tuyet Mai Thi To 1, Vladimir Kubyshkin 2, Franz-Josef Schmitt 3, Nediljko Budisa 1,2, Thomas Friedrich 1
1Institute of Chemistry, Technische Universität Berlin, 2Department of Chemistry, University of Manitoba, 3Institute of Physics, Martin-Luther-Universität Halle-Wittenberg

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

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