A bioreactor for in-cell protein NMR

Abstract

The inside of the cell is a complex environment that is difficult to simulate when studying proteins and other molecules in vitro. We have developed a device and system that provides a controlled environment for nuclear magnetic resonance (NMR) experiments involving living cells. Our device comprises two main parts, an NMR detection region and a circulation system. The flow of medium from the bottom of the device pushes alginate encapsulated cells into the circulation chamber. In the chamber, the exchange of oxygen and nutrients occurs between the media and the encapsulated cells. When the media flow is stopped, the encapsulated cells fall back into the NMR detection region, and spectra can be acquired. We have utilized the bioreactor to study the expression of the natively disordered protein alpha-synuclein, inside Escherichia coli cells.

Fig. 1

The CEC bioreactor. On the left, the pump is off and the encapsulates are settled. On the right, the pump is on and the encapsulates circulate at a steady state in the upper chamber. A: tubing inlet, B: NMR detection region, C: circulation chamber, D: adjustable threaded cap, E: fitting inlet. Orange circles represent encapsulates containing E. coli cells.

Fig. 2

The experimental set-up. A: Corning spinner flask fitted with a vented cap on one side arm and tubing adaptors on the other, B: peristaltic pump, C: water bath, D: 8 mm probe with heater removed, E: bioreactor, F: magnet, G: pH probe, H: computer, I: stir plate.

Fig. 3

Comparing α-synuclein spectra. A) In cell HSQC spectrum of alginate encapsulated E. coli expressing α-synuclein in the bioreactor. B) In cell HSQC spectrum of E. coli expressing α-synuclein. C) In vitro HSQC spectrum of 200 μM purified wild type α-synuclein in HEPES buffer, pH 7.2 at 10°C D) In cell HSQC spectrum of alginate encapsulated E. coli expressing α-synuclein. The spectra shown in panels A, B & D were acquired at 37°C. The spectra in panels B–D were acquired in a 5 mm NMR tube using a 5 mm probe. The spectrum in panel A was acquired in the 8 mm bioreactor using an 8 mm probe.

Fig. 4

In-cell SOFAST ¹⁵N-¹H HMQC [12] spectra (37°C) of E. coli expressing α-synuclein in the bioreactor. A) Spectrum collected before induction. B) 4 h post induction. C) 18 h post induction. D) Spectrum of the spent medium.

Fig. 5

Determining which crosspeaks correspond to α-synuclein. A) In-cell SOFAST ¹⁵N-¹H HMQC spectrum of the defined phosphate-free minimal media. B) Spectrum of ¹⁵N encriched encapsulated E. coli cells containing the control pUC18 plasmid. C) Spectrum of encapsulated E. coli expressing α-synuclein. D) Overlay of the spectra [medium (red), puc18 control cells (cyan), and α-synuclein (black)]. Crosspeaks used in subsequent analysis are labeled a-h. Spectra were acquired in the 8 mm bioreactor using an 8 mm probe at 37°C.

Fig. 6

Temporal changes in crosspeak volume after inducing α-synuclein expression in the bioreactor. A–E) α-Synuclein crosspeaks, F–G) Metabolite crosspeaks. H) Crosspeaks from the defined phosphate free minimal media. Crosspeak volumes are normalized to the largest volume and are labeled in Figure 5D. Error bars represent the standard error from three independent experiments.

Fig. 7

Schematic of the electrostatic encapsulation device. A: stir plate, B: beaker containing 150 mM CaCl₂, C: anigocatheter, D: needle, E: insulin syringe, F: alligator clip (connects the positive power supply terminal to the needle) G: high voltage power supply, H: ground, I: negative end