# Cell-free protein synthesis with technical additives – expanding the parameter space of in vitro gene expression

## TL;DR

This paper explores how adding various substances to a cell-free protein synthesis system can expand the conditions under which proteins can be made outside of cells.

## Contribution

The study systematically expands the parameter space of CFPS by testing ten technical additives under non-physiological conditions.

## Key findings

- The CFPS system is robust in synthesizing proteins even under high viscosities and altered osmolarity.
- Ten technical additives, including organic solvents and polymers, were tested with minimal negative impact on protein synthesis.
- Model proteins like sfGFP and thscGAS-sfGFP were successfully synthesized under diverse conditions.

## Abstract

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 machinery of an extract-donating organism to which substrates such as nucleotides and amino acids, as well as energy molecules, salts, buffer, etc., are added. After successful protein synthesis, further substrates can be added for an enzyme activity assay. Although mimicking of cell-like conditions is an approach for optimization, the physical and chemical properties of CFPS are not well described yet. To date, standard conditions have mainly been used for CFPS, with little systematic testing of whether conditions closer to intracellular conditions in terms of viscosity, macromolecules, inorganic ions, osmolarity, or water content are advantageous. Also, very few non-physiological conditions have been tested to date that would expand the parameter space in which CFPS can be performed. In this study, the properties of an Escherichia coli extract-based CFPS system are evaluated, and the parameter space is extended to high viscosities, concentrations of inorganic ion 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.