Automated production of functional membrane proteins using eukaryotic cell-free translation systems
| Autor: | Robert B. Quast, Stefan Kubick, Jörg Henkel, Marlitt Stech, Doreen A. Wüstenhagen, Oliver Kortt, Srujan Kumar Dondapati |
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| Přispěvatelé: | Publica |
| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
Azides
Lysis Potassium Channels Phenylalanine Recombinant Fusion Proteins KcsA potassium channel Bioengineering Biology Spodoptera Applied Microbiology and Biotechnology Amino Acyl-tRNA Synthetases Bacterial Proteins Protein biosynthesis Sf9 Cells Animals eukaryotic cell-free protein synthesis Integral membrane protein Luminescent Proteins automation chemistry.chemical_classification Aquaporin 1 General Medicine non-canonical amino acid electrophysiology Fusion protein Amino acid ErbB Receptors chemistry Membrane protein Biochemistry Bacteriorhodopsins integral membrane protein Heparin-binding EGF-like Growth Factor Plasmids Biotechnology fluorescence modification |
| Popis: | Due to their high abundance and pharmacological relevance there is a growing demand for the efficient production of functional membrane proteins. In this context, cell-free protein synthesis represents a valuable alternative that allows for the high-throughput synthesis of functional membrane proteins. Here, we demonstrate the potential of our cell-free protein synthesis system, based on lysates from cultured Spodoptera frugiperda 21 cells, to produce pro- and eukaryotic membrane proteins with individual topological characteristics in an automated fashion. Analytical techniques, including confocal laser scanning microscopy, fluorescence detection of eYFP fusion proteins in a microplate reader and in-gel fluorescence of statistically incorporated fluorescent amino acid derivatives were employed. The reproducibility of our automated synthesis approach is underlined by coefficients of variation below 7.2%. Moreover, the functionality of the cell-free synthesized potassium channel KcsA was analyzed electrophysiologically. Finally, we expanded our cell-free membrane protein synthesis system by an orthogonal tRNA/synthetase pair for the site-directed incorporation of p-Azido-l-phenylalanine based on stop codon suppression. Incorporation was optimized by performance of a two-dimensional screening with different Mg(2+) and lysate concentrations. Subsequently, the selective modification of membrane proteins with incorporated p-Azido-l-phenylalanine was exemplified by Staudinger ligation with a phosphine-based fluorescence dye. |
| Databáze: | OpenAIRE |
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