Synthesis and site-directed fluorescence labeling of azido proteins using eukaryotic cell-free orthogonal translation systems
| Autor: | Robert B. Quast, Helmut Merk, Iris Claussnitzer, Michael Gerrits, Stefan Kubick |
|---|---|
| Přispěvatelé: | Publica |
| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
Signal peptide
Azides Glycosylation Phenylalanine viruses Biophysics Biology Biochemistry Amino Acyl-tRNA Synthetases chemistry.chemical_compound Residue (chemistry) Sf9 Cells Animals Humans Erythropoietin Molecular Biology Triticum Fluorescent Dyes Glucuronidase chemistry.chemical_classification Cell-free protein synthesis Cell-Free System Vesicle Cell Biology Amino acid chemistry Microsome Electrophoresis Polyacrylamide Gel |
| Popis: | Eukaryotic cell-free systems based on wheat germ and Spodoptera frugiperda insect cells were equipped with an orthogonal amber suppressor tRNA–synthetase pair to synthesize proteins with a site-specifically incorporated p -azido- l -phenylalanine residue in order to provide their chemoselective fluorescence labeling with azide-reactive dyes by Staudinger ligation. The specificity of incorporation and bioorthogonality of labeling within complex reaction mixtures was shown by means of translation and fluorescence detection of two model proteins: β-glucuronidase and erythropoietin. The latter contained the azido amino acid in proximity to a signal peptide for membrane translocation into endogenous microsomal vesicles of the insect cell-based system. The results indicate a stoichiometric incorporation of the azido amino acid at the desired position within the proteins. Moreover, the compatibility of cotranslational protein translocation, including glycosylation and amber suppression-based incorporation of p -azido- l -phenylalanine within a cell-free system, is demonstrated. The presented approach should be particularly useful for providing eukaryotic and membrane-associated proteins for investigation by fluorescence-based techniques. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|