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

• Tabea Bartsch,
• Stephan Lütz and
• Katrin Rosenthal

Beilstein J. Org. Chem. 2024, 20, 2242–2253, doi:10.3762/bjoc.20.192

• 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; cGAS; Escherichia coli cell-free extract; sfGFP; TX-TL; Introduction In addition to other applications such as biomanufacturing or biosensing, cell-free protein synthesis (CFPS) of enzymes has established itself as a tool for rapid screening of

• properties beyond physiological ranges. We also tested several organic solvents that are miscible and non-miscible with water. For the experiments, two model proteins, the superfolder GFP (sfGFP) and the enzyme human cyclic GMP-AMP synthase-sfGFP, were used, which differ in their size (sfGFP: 27 kDa, thscGAS