Cell-free protein synthesis of perdeuterated proteins for NMR studies |
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Authors: | Touraj Etezady-Esfarjani Sebastian Hiller Cristina Villalba Kurt Wüthrich |
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Affiliation: | (1) Institute for Molecular Biology and Biophysics, ETH Zurich, Zurich, 8093, Switzerland;(2) Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA |
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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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Keywords: | Back protonation of deuterated amino acids Cell-free protein synthesis Deuterated proteins GroEL Isotope labeling |
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