Cell-Free Protein Synthesis with Technical Additives – Expanding the Parameter Space of In Vitro Gene Expression

Submitting author affiliation:
Constructor University, Bremen, Germany

Beilstein Arch. 2024, 202441. https://doi.org/10.3762/bxiv.2024.41.v1

Published 13 Jun 2024

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This preprint has not been peer-reviewed. When a peer-reviewed version is available, this information will be updated.


Biocatalysis has established itself as a successful tool in organic synthesis. A particularly fast technique for screening enzymes is the in vitro expression or cell-free protein synthesis (CFPS). The system is based on the transcription and translation system of an extract donating organism to which substrates like nucleotides and amino acids, as well as energy molecules, salts, buffer, etc. have to be added. After successful protein synthesis, further substrates can be added for an enzyme activity assay. mimicking of cell like conditions is one approach for optimization, the physical and chemical properties of CFPS are not well described yet. To date, mainly standard conditions have been used for CFPS, with little systematic testing of whether conditions closer to intracellular conditions with regards to viscosity, macromolecules, inorganic ions, osmolarity, or water content are advantageous. Also, no non-physiological conditions have been tested to date that would expand the parameter space in which CFPS can be performed. In this study, properties of an Escherichia coli extract based CFPS system are evaluated, and the parameter space is expanded to high viscosities, concentrations of inorganic ions and osmolarity using ten different technical additives including organic solvents, polymers, and salts. It is shown that the synthesis of two model proteins, namely superfolder GFP (sfGFP) and the enzyme truncated human cyclic GMP-AMP synthase fused to sfGFP (thscGAS-sfGFP), is very robust against most of the tested additives.

Keywords: Cell-free protein synthesis; Escherichia coli cell-free extract; TXTL; sfGFP; cGAS

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When a peer-reviewed version of this preprint is available, this information will be updated in the information box above. If no peer-reviewed version is available, please cite this preprint using the following information:

Bartsch, T.; Lütz, S.; Rosenthal, K. Beilstein Arch. 2024, 202441. doi:10.3762/bxiv.2024.41.v1

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