The transient expression of recombinant proteins in plant cell packs facilitates stable isotope labelling for NMR spectroscopy

Abstract

Nuclear magnetic resonance (NMR) spectroscopy can be used to determine the structure, dynamics and interactions of proteins. However, protein NMR requires stable isotope labelling for signal detection. The cells used for the production of recombinant proteins must therefore be grown in medium containing isotopically labelled substrates. Stable isotope labelling is well established in Escherichia coli, but bacteria are only suitable for the production of simple proteins without post-translational modifications. More complex proteins require eukaryotic production hosts, but their growth can be impaired by labelled media, thus reducing product yields and increasing costs. To address this limitation, we used media supplemented with isotope-labelled substrates to cultivate the tobacco-derived cell line BY-2, which was then cast into plant cell packs (PCPs) for the transient expression of a labelled version of the model protein GB1. Mass spectrometry confirmed the feasibility of isotope labelling with ¹⁵ N and ² H using this approach. The resulting NMR spectrum featured a signal dispersion comparable to recombinant GB1 produced in E. coli. PCPs therefore offer a rapid and cost-efficient alternative for the production of isotope-labelled proteins for NMR analysis, especially suitable for complex proteins that cannot be produced in microbial systems.

Keywords: alternative expression host; defined cultivation media; isotope labelling; plant cell culture; structural analysis.

Figure 1

BY‐2 cell viability in labelled and control media. (a) A gradually increasing volume fraction of deuterium oxide reduced cell viability over time. Cell passaging times are indicated by dashed vertical lines. Numbers in the legend indicate the percentage deuterium oxide in the medium at the start of each passage v/v, for example 20–50–50 corresponds to cells cultured in 20% D2O in passage 1 and then at 50% D2O in passages 2 and 3. (b) The correlation between the deuterium oxide volume fraction and loss of cell viability was linear (Pearson's r = −0.991, adj. R ² = 0.986) if the water control was excluded, indicating a deuterium oxide no‐effect volume fraction of ~11% v/v (red dot). (c) BY‐2 cells in media containing different labelled components and control medium. Cells in 50% v/v deuterium oxide had been passaged with this volume fraction ~27 days before starting the cultivation shown here (see panel a, 20–50–50 regime). Details on the cell properties at the time of harvest are given in Table 1. (d) Microscopic images of BY‐2 cells cultivated in labelled and control media after staining with Evans blue. Scale bar = 50 μm. H—control medium, regular water; D—medium prepared using 50% v/v deuterium oxide; N—medium prepared using regular water but containing labelled ammonium nitrate (15NH415NO3); DN—medium prepared using 50% v/v deuterium oxide and labelled ammonium nitrate. Error bars in a–c indicate the standard deviation (n ≥ 2). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Expression of plastid‐targeted GB1 in PCPs prepared from BY‐2 cells grown in labelled and control media, and purification from PCP extracts by immobilized metal affinity chromatography (IMAC). (a) Accumulation of GB1 in BY‐2 PCPs cultivated in media with (D, N, DN) and without (H, control) isotopes was estimated from dot blots using a rabbit anti‐His₆ primary antibody and an alkaline phosphatase‐labelled goat anti‐rabbit secondary antibody. GB1 was quantified by densitometry against an authentic GB1 standard in eight dilutions. Error bars show standard deviations from 12–14 PCPs (***P < 0.0001; **P < 0.01). (b) Accumulation of GB1 and DsRed in plastids of PCPs prepared from BY‐2 cells grown in labelled media. DsRed fluorescence was quantified using authentic standards and GB1 was quantified by the densitometric analysis of dot blots against corresponding GB1 standards. The correlation between DsRed and GB1 was linear (Pearson's r = 0.980, R ² = 0.961, Adj. R ² = 0.921). The error bars for GB1 are considerably larger than those for DsRed because the densitometric quantitation of the former was less precise than fluorescence‐based measurements for DsRed. D—medium prepared using 50% v/v deuterium oxide; N—medium prepared using regular water but containing labelled ammonium nitrate (¹⁵NH₄ ¹⁵NO₃); DN—medium prepared using 50% v/v deuterium oxide and labelled ammonium nitrate. (c and d) Samples collected during the purification of plastid‐targeted GB1 by IMAC were analysed by LDS gel electrophoresis and subsequent staining with Coomassie Brilliant Blue (c) and Western blotting (d) using the same antibodies as for the dot blot (see panel a). Black arrows indicate the expected size of GB1. S—DsRed standard (10 μg/mL), M—marker, H—PCP extract, FT—IMAC flow through, WIMAC wash (30 mm imidazole), E1–E3—IMAC elution fractions. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

ESI‐MS and NMR analysis of GB1 samples derived from plant cells. (a) Amino acid sequence of GB1 after the proteolytic removal of the signal peptide. (b) Mass spectra of the intact protein from the four GB1 samples produced in different isotopic media. The molecular mass of the GB1 species is indicated above the spectrum in each case and the corresponding degree of isotope labelling is shown in Table 3. (c) Overlay of the 2D‐[¹⁵N,¹H]‐HSQC spectra of the ²H,¹⁵N‐labelled GB1 plant‐derived sample (medium DN, plastid‐targeted, blue) and ²H,¹⁵N‐labelled GB1 produced in E. coli (red). Sequence‐specific resonance assignments are labelled (Ikeya et al.,  2016). (d) Chemical shift perturbation (CSP) plot indicating only minor spectral changes between the two samples in c. (e) Significant CSPs mapped onto the structure of GB1 (2n9k.pdb (Ikeya et al.,  2016)) indicates slight changes at the N‐terminal end of the protein. [Colour figure can be viewed at wileyonlinelibrary.com]