In-cell protein NMR and protein leakage

Abstract

In-cell nuclear magnetic resonance spectroscopy is a tool for studying proteins under physiologically relevant conditions. In some instances, however, protein signals from leaked protein are observed in the liquid surrounding the cells. Here, we examine the expression of four proteins in Escherichia coli. We describe the controls that should be used for in-cell NMR experiments and show that leakage is likely when the protein being studied exceeds ∼20% of the total cellular protein.

Figure 1

SOFAST ¹⁵N – ¹H HMQC spectra from E. coli expressing HdeA after 3.0 h of induction [A, cell slurry; B, supernatant immediately after acquisition of cell slurry spectrum; C, cell lysate].

Figure 2

Spectra from E. coli expressing HdeA after 1.5 h of induction. The panels are described in the legend to Figure 1.

Figure 3

Spectra from E. coli expressing CI2 after induction for 1.5 h (left panels) and 3.0 h (right panels) [A–B, cell slurry; C–D, supernatant immediately after acquisition of cell slurry spectra; E–F, cell lysates].