Abstract
Cell-free protein synthesis protocols for uniformly deuterated proteins typically yield low, non-uniform deuteration levels. This paper introduces an E. coli cell-extract, D-S30, which enables efficient production of proteins with high deuteration levels for all non-labile hydrogen atom positions. Potential applications of the new protocol may include production of proteins with selective isotope-labeling of selected amino acid residues on a perdeuterated background for studies of enzyme active sites or for ligand screening in drug discovery projects, as well as the synthesis of perdeuterated polypeptides for NMR spectroscopy with large supra-molecular structures. As an illustration, it is demonstrated that the 800-kDa chaperonine GroEL synthesized with the D-S30 cell-free system had a uniform deuteration level of about 95% and assembled into its biologically active oligomeric form.
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Abbreviations
- CECF:
-
continuous-exchange cell-free
- FKBP:
-
FK506-binding protein
- MS:
-
mass spectrometry
- MWCO:
-
molecular weight cut-off
- RNAP:
-
RNA polymerase.
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Acknowledgements
We thank Dr. A. Strauss, Protein Structure Unit of the Novartis Biomedical Research Institute, Basel for providing us with the FKBP expression plasmid. The deuterated amino acid mixture used in this work was a generous gift from Cambridge Isotope Laboratories, Inc. (CIL). Financial support was obtained from the Schweizerischer Nationalfonds and the ETH Zürich through the NCCR Structural Biology.
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Etezady-Esfarjani, T., Hiller, S., Villalba, C. et al. Cell-free protein synthesis of perdeuterated proteins for NMR studies . J Biomol NMR 39, 229–238 (2007). https://doi.org/10.1007/s10858-007-9188-0
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DOI: https://doi.org/10.1007/s10858-007-9188-0