Jan 12, 1999

Predictable deuteration of recombinant proteins expressed in Escherichia coli

Analytical Biochemistry
B LeitingJ F O'Connell

Abstract

Random deuteration of recombinant proteins in Escherichia coli is widely used for protein structure determination by nuclear magnetic resonance (NMR). It is desirable to predict accurately the degree of deuteration because each NMR experiment benefits from a different level of deuteration. The method described here which uses [2H]2O and glucose as the sole carbon and deuterium sources is an alternative for a previously published procedure using acetate and [2H]2O (Venter et al., J. Biomol. NMR 5, 339-344, 1995) and it is of advantage for proteins that do not express well using acetate. While the deuteration degree with acetate is approximately linear with the [2H]2O content in the medium, the use of glucose leads to deviations up to 19%, which is analyzed systematically here. With [2H]2O as the sole deuterium source 0-86% of the chemically nonexchangeable hydrogen atoms can be deuterated. Higher levels of deuteration require perdeuterated glucose in combination with [2H]2O. As an example, recombinant peptide deformylase from Bacillus subtilis was overexpressed, deuterated to various degrees, purified, and analyzed by mass spectrometry and NMR.

Mentioned in this Paper

In Vivo NMR Spectroscopy
Alkalescens-Dispar Group
Peptide deformylase
Magnetic Resonance Imaging
Proteins, Recombinant DNA
Def protein, E coli
Aminopeptidase
Mass Spectrometry
Bacillus subtilis
Oligonucleotide Primers

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