A simple protocol for the production of highly deuterated proteins for biophysical studies.
J Biol Chem
; 298(8): 102253, 2022 08.
Article
en En
| MEDLINE
| ID: mdl-35835218
ABSTRACT
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 (2H2O) oxide M9 medium compared with that in 1H2O M9 medium. The protocol will enable a broader utilization of deuterated proteins in a number of biophysical techniques.
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Banco de datos:
MEDLINE
Asunto principal:
Biofisica
/
Proteínas Recombinantes
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Deuterio
Idioma:
En
Año:
2022
Tipo del documento:
Article