Predictable deuteration of recombinant proteins expressed in Escherichia coli.
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.
Leiting
B
B
Department of Biochemistry, Merck Research Laboratories, Rahway, New Jersey, 07065, USA. Barbara_Leiting@Merck.com
Marsilio
F
F
O'Connell
J F
JF
D000581:Amidohydrolases; D000626:Aminopeptidases / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D003903:Deuterium / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D017931:DNA Primers; D011994:Recombinant Proteins; D003903:Deuterium; D000626:Aminopeptidases; D000581:Amidohydrolases; C084507:peptide deformylase