Preprints
https://doi.org/10.5194/mr-2023-3
https://doi.org/10.5194/mr-2023-3
15 Mar 2023
 | 15 Mar 2023
Status: this preprint is currently under review for the journal MR.

Cell-free Synthesis of Proteins with Selectively 13C-Labelled Methyl Groups from Inexpensive Precursors

Damian Van Raad, Gottfried Otting, and 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.

Damian Van Raad et al.

Status: open (until 12 Apr 2023)

Comment types: AC – author | RC – referee | CC – community | EC – editor | CEC – chief editor | : Report abuse
  • CC1: 'Comment on mr-2023-3', Anja Böckmann, 15 Mar 2023 reply
  • RC1: 'Comment on mr-2023-3', Anja Böckmann, 21 Mar 2023 reply
  • RC2: 'Comment on mr-2023-3', Anonymous Referee #2, 29 Mar 2023 reply

Damian Van Raad et al.

Data sets

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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Short summary
A novel cell-free protein synthesis system called eCells produces amino acids based on specific isotopes using low-cost precursors. The system selectively labels methyl groups; i.e valine and leucine with high efficiency. eCells achieve high levels of 13C incorporation and deuteration in protein preparations, making them suitable for NMR experiments of large protein complexes. It is easy to prepare and can be scaled up in volume, making it a promising tool for protein production and NMR studies.