Improving cell-free expression of membrane proteins by tuning ribosome co-translational membrane association and nascent chain aggregation

Abstract

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.

Evaluation of the Ion Channel Assembly in a Eukaryotic Cell-Free System Focusing on Two-Pore Domain Potassium Channels K2P

“…These results correspond to the previous time series ( Figure 2 b) and additionally suggest the requirement of further protein processing time for higher order oligomer assembly. It should be noted that the absence of microsomes reduced the protein yield, consistent with other studies [ 23 ] and thus causes overall weaker protein bands in the autoradiogram.…”

Engineering transmembrane signal transduction in synthetic membranes using two-component systems

“…3 C ). This nitrate-independent increase in luminescence upon addition of POPE may suggest that negative lipid curvature stabilizes the “on” conformation of NarX ( 44 ). The high induction of luciferase expression in POPE and POPG membranes is unsurprising as PE and PG make up the majority of lipid headgroups in E. coli membranes ( 46 ).…”

“…S5 ). Further, improper membrane protein folding has been found to increase aggregation and truncation products ( 41 , 44 ), and likely inhibits efficient expression of all protein components in the system. As a result of this relationship, we only considered NarX expression in our future analysis of the effect of membrane composition on NarX-L performance.…”

“…However, luminescence for the vancomycin sensor was highest across all conditions ( Fig. 4 E ), suggesting that the chimera may be locked into an on conformation, perhaps due to destabilizing the HAMP domain ( 44 ). These results demonstrate how protein and membrane engineering can be used to readily generate cell-free receptors by tapping into the large repertoire of known sensing domains of transmembrane proteins, and underscores the important relationship between membrane physical properties, protein sequence, and function.…”

Cell-free expression of Nipah virus transmembrane proteins for proteoliposome vaccine design

“…However, this peptide is unnecessary in our cell-free system that lacks a signal recognition particle and we hypothesized that the presence of the hydrophobic peptide in the nascent peptide chain might hinder the cotranslational integration of the membrane protein into liposome membranes. Further, we have previously found that sequence alterations in the nascent chain of a cell-free expressed membrane protein can have a profound impact on cell-free membrane protein expression (56). Specifically, we identified that alterations in protein sequence that inhibit protein-lipid interactions lead to membrane proteins that cannot successfully move off the ribosome and insert into membranes.…”