This title appears in the Scientific Report :
2017
Please use the identifier:
http://dx.doi.org/10.1038/srep46753 in citations.
Please use the identifier: http://hdl.handle.net/2128/14259 in citations.
A Novel Method to Evaluate Ribosomal Performance in Cell-Free Protein Synthesis Systems1038/srep
A Novel Method to Evaluate Ribosomal Performance in Cell-Free Protein Synthesis Systems1038/srep
Cell-free protein synthesis (CFPS) systems were designed to produce proteins with a minimal set of purified components, thus offering the possibility to follow translation as well as protein folding. In order to characterize the performance of the ribosomes in such a system, it is crucial to separat...
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Personal Name(s): | Kempf, Noemie |
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Remes, Cristina / Ledesch, Ralph / Züchner, Tina / Höfig, Henning / Ritter, Ilona / Katranidis, Alexandros (Corresponding author) / Fitter, Jörg | |
Contributing Institute: |
Molekulare Biophysik; ICS-5 |
Published in: | Scientific reports, 7 (2017) S. 46753 |
Imprint: |
London
Nature Publishing Group
2017
|
PubMed ID: |
28436469 |
DOI: |
10.1038/srep46753 |
Document Type: |
Journal Article |
Research Program: |
Functional Macromolecules and Complexes |
Link: |
OpenAccess OpenAccess |
Publikationsportal JuSER |
Please use the identifier: http://hdl.handle.net/2128/14259 in citations.
Cell-free protein synthesis (CFPS) systems were designed to produce proteins with a minimal set of purified components, thus offering the possibility to follow translation as well as protein folding. In order to characterize the performance of the ribosomes in such a system, it is crucial to separately quantify the two main components of productivity, namely the fraction of active ribosomes and the number of synthesizing cycles. Here, we provide a direct and highly reliable measure of ribosomal activity in any given CFPS system, introducing an enhanced-arrest peptide variant. We observe an almost complete stalling of ribosomes that produce GFPem (~95%), as determined by common centrifugation techniques and fluorescence correlation spectroscopy (FCS). Moreover, we thoroughly study the effect of different ribosomal modifications independently on activity and number of synthesizing cycles. Finally, employing two-colour coincidence detection and two-colour colocalisation microscopy, we demonstrate real-time access to key productivity parameters with minimal sample consumption on a single ribosome level. |