Abstract
The in-cell NMR technique offers significant insights into the structure and function of heterologous proteins in the physiological intracellular environment at an atomic resolution. Escherichia coli (E. coli) is one of the most widely used host cells for heterologous protein expression in structural biological studies as well as for in-cell NMR studies to investigate fundamental structural characteristics and the physiochemistry of certain proteins and their intermolecular interactions under physiological conditions. However, in many cases, it is not easy to obtain well-resolved in-cell NMR spectra because the detectability and resolution of these spectra are significantly influenced by intracellular factors such as nonspecific intermolecular interactions. In this study, we re-examined the experimental parameters of E. coli in-cell NMR and found that the detectability and resolution of the NMR spectra clearly depended on the growth phase of the host cells. Furthermore, the detectability and resolution of the E. coli in-cell NMR spectra correlated with the soluble fraction amounts of the expressed target protein. These results indicate that the E. coli in-cell NMR spectrum of a target protein is a useful tool for monitoring the intracellular conditions of the host cell and for establishing the appropriate cultivation conditions for protein overexpression.
Highlights
Solution NMR spectroscopy is nondestructive and is widely used as a tool for the in vivo observation of metabolites and metal ions in living cells, for which it is ideal[1]
Escherichia coli (E. coli) is one of the most widely used host cells for the overexpression of heterologous proteins, as well as for in-cell protein NMR studies to elucidate fundamental structural and physicochemical characteristics of proteins and their intermolecular interaction modes under physiological conditions[8,9,10,11,12,13,14]
The detectability and resolution of E. coli in-cell NMR spectra of GB1 drastically depended on the OD600 value at the time of the addition of isopropyl β-d-1-thiogalactopyranoside (IPTG), and this parameter was the most critical to improve in-cell NMR spectra (Supplemental Fig. S1)
Summary
Solution NMR spectroscopy is nondestructive and is widely used as a tool for the in vivo observation of metabolites and metal ions in living cells, for which it is ideal[1]. The detectability and resolution of E. coli in-cell NMR spectra of GB1 drastically depended on the OD600 value at the time of the addition of IPTG, and this parameter was the most critical to improve in-cell NMR spectra (Supplemental Fig. S1).
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