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A simple protocol for the production of highly deuterated proteins for biophysical studies

Highly deuterated protein samples expand the biophysics and biological tool kit by providing, among other qualities, contrast matching in neutron diffraction experiments and reduction of dipolar spin interactions from normally protonated proteins in magnetic resonance studies, impacting both electro...

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Detalles Bibliográficos
Autores principales: Li, Jess, Byrd, R. Andrew
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9386462/
https://www.ncbi.nlm.nih.gov/pubmed/35835218
http://dx.doi.org/10.1016/j.jbc.2022.102253
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author Li, Jess
Byrd, R. Andrew
author_facet Li, Jess
Byrd, R. Andrew
author_sort Li, Jess
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description Highly deuterated protein samples expand the biophysics and biological tool kit by providing, among other qualities, contrast matching in neutron diffraction experiments and reduction of dipolar spin interactions from normally protonated proteins in magnetic resonance studies, impacting both electron paramagnetic resonance and NMR spectroscopy. In NMR applications, deuteration is often combined with other isotopic labeling patterns to expand the range of conventional NMR spectroscopy research in both solution and solid-state conditions. However, preparation of deuterated proteins is challenging. We present here a simple, effective, and user-friendly protocol to produce highly deuterated proteins in Escherichia coli cells. The protocol utilizes the common shaker flask growth method and the well-known pET system (which provides expression control via the T7 promotor) for large-scale recombinant protein expression. One liter expression typically yields 5 to 50 mg of highly deuterated protein. Our data demonstrate that the optimized procedure produces a comparable quantity of protein in deuterium ((2)H(2)O) oxide M9 medium compared with that in (1)H(2)O M9 medium. The protocol will enable a broader utilization of deuterated proteins in a number of biophysical techniques.
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spelling pubmed-93864622022-08-22 A simple protocol for the production of highly deuterated proteins for biophysical studies Li, Jess Byrd, R. Andrew J Biol Chem Methods and Resources Highly deuterated protein samples expand the biophysics and biological tool kit by providing, among other qualities, contrast matching in neutron diffraction experiments and reduction of dipolar spin interactions from normally protonated proteins in magnetic resonance studies, impacting both electron paramagnetic resonance and NMR spectroscopy. In NMR applications, deuteration is often combined with other isotopic labeling patterns to expand the range of conventional NMR spectroscopy research in both solution and solid-state conditions. However, preparation of deuterated proteins is challenging. We present here a simple, effective, and user-friendly protocol to produce highly deuterated proteins in Escherichia coli cells. The protocol utilizes the common shaker flask growth method and the well-known pET system (which provides expression control via the T7 promotor) for large-scale recombinant protein expression. One liter expression typically yields 5 to 50 mg of highly deuterated protein. Our data demonstrate that the optimized procedure produces a comparable quantity of protein in deuterium ((2)H(2)O) oxide M9 medium compared with that in (1)H(2)O M9 medium. The protocol will enable a broader utilization of deuterated proteins in a number of biophysical techniques. American Society for Biochemistry and Molecular Biology 2022-07-12 /pmc/articles/PMC9386462/ /pubmed/35835218 http://dx.doi.org/10.1016/j.jbc.2022.102253 Text en https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Methods and Resources
Li, Jess
Byrd, R. Andrew
A simple protocol for the production of highly deuterated proteins for biophysical studies
title A simple protocol for the production of highly deuterated proteins for biophysical studies
title_full A simple protocol for the production of highly deuterated proteins for biophysical studies
title_fullStr A simple protocol for the production of highly deuterated proteins for biophysical studies
title_full_unstemmed A simple protocol for the production of highly deuterated proteins for biophysical studies
title_short A simple protocol for the production of highly deuterated proteins for biophysical studies
title_sort simple protocol for the production of highly deuterated proteins for biophysical studies
topic Methods and Resources
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9386462/
https://www.ncbi.nlm.nih.gov/pubmed/35835218
http://dx.doi.org/10.1016/j.jbc.2022.102253
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