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

Abstract

Summary 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 15N and 2H 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.

Multi-omics revolution to promote plant breeding efficiency

“…Proteomics is a technique for analyzing all the proteins expressed within an organism, and it splits into four major types: sequence, structure, function, and expression proteomics ( Aizat and Hassan, 2018 ). Different approaches are used to analyze these types, for intence, sequence proteomics analyzed by HPLC (high-performance liquid chromatography) ( Hao et al., 2018 ) and structural proteomics by nuclear magnetic resonance (NMR), electron microscopy, crystallization, and X-ray diffraction of protein crystals ( Woolfson, 2018 ; Opdensteinen et al., 2022 ). However, functional proteomics is examined through various methods as yeast one (Y1H) or two hybrids (Y2H) and protein micro-array profiling ( Yang et al., 2020 ; Mao et al., 2020 ).…”

Reducing water uptake into BY‐2 cells by systematically optimizing the cultivation parameters increases product yields achieved by transient expression in plant cell packs

“…[ 23 ] All enzymes for cloning were provided by New England Biolabs (Ipswich, USA). We also tested the expression of GB1, [ 18 ] DsRed, [ 13 ] and our previously described IgG1 and IgG3 antibodies. [ 24 ]…”

“…The fraction of living cells was determined by Evans blue staining [ 26 ] using a trinocular microscope (BMS, Breukhoven, Netherlands) equipped with a 40× objective and a 10× eyepiece as previously described. [ 18 ] A Fuchs‐Rosenthal chamber was used to count cells for growth curve analysis and to calculate the relative cell wet and dry masses. When investigating BY‐2 growth curves, multiple flasks were cultivated in parallel and terminated after sampling to prevent any potential lag resulting from repeated sampling.…”

“…[ 17 ] PCP technology would also greatly benefit from an overall increase in expression levels so that a typical ∼60 mg PCP can provide sufficient protein for initial analysis and functional testing, such as 1 mg required for nuclear magnetic resonance spectroscopy. [ 18 ]…”

“…[17] PCP technology would also greatly benefit from an overall increase in expression levels so that a typical ∼60 mg PCP can provide sufficient protein for initial analysis and functional testing, such as 1 mg required for nuclear magnetic resonance spectroscopy. [18] Here, we systematically investigated the BY-2 cultivation parameters and PCP preparation steps that affect batch-to-batch variation and protein accumulation in PCPs. We applied a design of experiments (DoE) approach to investigate the effects of antifoam additives, carbon sources, osmolality and pH in the medium, BY-2 and PCP harvest time points, and exposure to light.…”