Accedi

Technical University of Berlin

5 ARTICLES PUBLISHED IN JoVE

Biology

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Katharina L. Dürr^1,2, Neslihan N. Tavraz¹, Susan Spiller¹, Thomas Friedrich¹

¹Institute of Chemistry, Technical University of Berlin, ²The Vollum Institute, Oregon Health & Science University

We describe a method to quantify the activity of K⁺-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb⁺ or Li⁺ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

Biology

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

Emanuel G. Worst¹, Matthias P. Exner², Alessandro De Simone², Marc Schenkelberger¹, Vincent Noireaux³, Nediljko Budisa², Albrecht Ott¹

¹Department of Experimental Physics, Saarland University, ²Institute of Chemistry, Technische Universität Berlin, ³School of Physics and Astronomy, University of Minnesota

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

Bioengineering

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Tobias Baumann¹, Franz-Josef Schmitt², Almut Pelzer¹, Vivian Jeanette Spiering³, Georg Johannes Freiherr von Sass¹, Thomas Friedrich², Nediljko Budisa¹

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).

Bioengineering

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Jessica H. Nickling^*1, Tobias Baumann^*1, Franz-Josef Schmitt², Maike Bartholomae³, Oscar P. Kuipers³, Thomas Friedrich², Nediljko Budisa¹

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.

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 To¹, Vladimir Kubyshkin², Franz-Josef Schmitt³, Nediljko Budisa^1,2, Thomas Friedrich¹

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.