PT - JOURNAL ARTICLE AU - Steinküher, Jan AU - Peruzzi, Justin A. AU - Krüger, Antje AU - Jacobs, Miranda L. AU - Jewett, Michael C. AU - Kamat, Neha P. TI - Improving cell-free expression of membrane proteins by tuning ribosome co-translational membrane association and nascent chain aggregation AID - 10.1101/2023.02.10.527944 DP - 2023 Jan 01 TA - bioRxiv PG - 2023.02.10.527944 4099 - http://biorxiv.org/content/early/2023/02/10/2023.02.10.527944.short 4100 - http://biorxiv.org/content/early/2023/02/10/2023.02.10.527944.full AB - Cell-free gene expression (CFE) systems are powerful tools for transcribing and translating genes outside of a living cell. Given their diverse roles in nature, synthesis of membrane proteins is of particular interest, but their yield in CFE is substantially lower than for soluble protein. In this paper, we study factors that affect the cell-free synthesis of membrane proteins and develop a quantitative kinetic model of their production. We identify that stalling of membrane protein translation on the ribosome is a strong predictor of membrane protein synthesis and creates a negative feedback loop in which stalled peptide sequences quench ribosome activity through aggregation between the ribosome nascent chains. Synthesis can be improved by the addition of lipid membranes which incorporate protein nascent chains and, therefore, kinetically competes with aggregation. Using both quantitative modeling and experiment, we show that the balance between peptide-membrane association and peptide aggregation rates determines the total yield of synthesized membrane protein. We then demonstrate that this balance can be shifted by altering membrane composition or the protein N-terminal domain sequence. Based on these findings, we define a membrane protein expression score that can be used to rationalize the engineering of N-terminal domain sequences both of a native and computationally designed membrane proteins produced through CFE.Competing Interest StatementThe authors have declared no competing interest.