the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
Cell-free Synthesis of Proteins with Selectively 13C-Labelled Methyl Groups from Inexpensive Precursors
Damian Raad
Gottfried Otting
Thomas Huber
Abstract. The novel eCell system maintains the activity of the entire repertoire of metabolic E. coli enzymes in cell-free protein synthesis. We show that this can be harnessed to produce proteins with selectively 13C-labelled amino acids from inexpensive 13C-labelled precursors. The system is demonstrated with selective 13C-labelling of methyl groups in the proteins ubiquitin and peptidyl-prolyl cis–trans isomerase B. Starting from 3-13C-pyruvate, 13C-HSQC cross-peaks are obtained devoid of one-bond 13C-13C scalar couplings. Starting from 2-13C-methyl-acetolactate, single methyl groups of valine and leucine are labelled. Labelling efficiencies are 70 % or higher, and the method allows to produce perdeuterated proteins with protonated methyl groups in residue-selective manner. The system uses the isotope-labelled precursors sparingly and is more readily scalable than conventional cell-free systems.
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Damian Van Raad et al.
Status: open (until 12 Apr 2023)
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CC1: 'Comment on mr-2023-3', Anja Böckmann, 15 Mar 2023
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This is an excellent and important paper, very carfeully written, fully complete with all necessary info. It can be published as is.Â
Citation: https://doi.org/10.5194/mr-2023-3-CC1 -
RC1: 'Comment on mr-2023-3', Anja Böckmann, 21 Mar 2023
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This is an excellent and important paper, very carfeully written, fully complete with all necessary info. It can be published as is.Â
Citation: https://doi.org/10.5194/mr-2023-3-RC1 -
RC2: 'Comment on mr-2023-3', Anonymous Referee #2, 29 Mar 2023
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In this paper, the authors present a cell-free approach to produce proteins selectively labeled with 13CH3 groups using inexpensive precursors. This is an innovative method that will be an interesting alternative for labeling large proteins for solution NMR studies. The article is well written, the data are convincing. The reviewer recommends publication after the following revisions:
- Methyl-specific labeling of proteins in vitro using 2-keto acids has been previously reported in the literature (Lazarova et al, ACS Chem. Biol. 2018, 13, 2170-2178), including the standard 2-ketoisovalerate for valine labeling. This should be clearly stated in the introduction and commented in the discussion section.
- The most useful methyl probes in large proteins are ile-d1. Can the proposed approach be used with standard 2-ketobutyrate for specific labeling of ile-d1 methyl groups? The papers will clearly be improved if these data are included.
- Methyl-specific labeling is very important for the NMR studies of very large proteins, but it requires the simultaneous perdeuteration of proteins. In vitro, this requires the use of perdeuterated amino acids at a concentration of 1 mM for each amino acid in addition to the methyl-labeled amino acids or precursors. Reconstitution of these mixtures can be expensive and the cost of the 2H amino acids used should be considered in the cost estimates in Table 1 and in the discussion section.
- Lines 34-35: "Alternatively, isotope-labeled precursors can be provided, but this generally does not solve the problem of 13C-13C couplings." This sentence is inaccurate: standard 2-ketobutyrate, 2-ketoisovalerate, and acetolactate are all commercially available and used in standard applications, in forms where only one carbon is specifically labeled, which solves the 13C-13C coupling problem.
- Line 69: please indicate that 2-ketobutyrate is used for isotopic labeling of Ile-d1 methyl groups
- Line 247: please state why perdeuterated pyruvate was used in combination with acetolactate to produce a sample specifically labeled on the Pro-S methyl groups.
- Figures 3 and 4: please add examples of 1D traces at appropriate locations to show whether or not there is residual labeling for valine, leucine or pro-R methyl groups.
Citation: https://doi.org/10.5194/mr-2023-3-RC2
Damian Van Raad et al.
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Cell-free Synthesis of Proteins with Selectively 13C-Labelled Methyl Groups from Inexpensive Precursors- Dataset Damian Van Raad, Gottfried Otting, and Thomas Huber https://zenodo.org/record/7662927
Damian Van Raad et al.
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