Automated production of functional membrane proteins using eukaryotic cell-free translation systems

Abstract

Due to their high abundance and pharmacological relevance there is a growing demand for the efficient production of functional membrane proteins. In this context, cell-free protein synthesis represents a valuable alternative that allows for the high-throughput synthesis of functional membrane proteins. Here, we demonstrate the potential of our cell-free protein synthesis system, based on lysates from cultured Spodoptera frugiperda 21 cells, to produce pro- and eukaryotic membrane proteins with individual topological characteristics in an automated fashion. Analytical techniques, including confocal laser scanning microscopy, fluorescence detection of eYFP fusion proteins in a microplate reader and in-gel fluorescence of statistically incorporated fluorescent amino acid derivatives were employed. The reproducibility of our automated synthesis approach is underlined by coefficients of variation below 7.2%. Moreover, the functionality of the cell-free synthesized potassium channel KcsA was analyzed electrophysiologically. Finally, we expanded our cell-free membrane protein synthesis system by an orthogonal tRNA/synthetase pair for the site-directed incorporation of p-Azido-l-phenylalanine based on stop codon suppression. Incorporation was optimized by performance of a two-dimensional screening with different Mg(2+) and lysate concentrations. Subsequently, the selective modification of membrane proteins with incorporated p-Azido-l-phenylalanine was exemplified by Staudinger ligation with a phosphine-based fluorescence dye.

Production of G protein‐coupled receptors in an insect‐based cell‐free system

“…The effect of the melittin signal sequence is not fully understood. According to previous publications, demonstrating that the melittin signal sequence enhanced the translocation of secreted and type‐I‐transmembrane proteins into microsomes (Quast et al, ), it can be assumed that this effect might also account for certain cell‐free synthesized GPCRs. In addition, fusion of a signal peptide (melittin signal sequence) to the target gene could have positive influence on the synthesis and orientation of the GPCR in the vesicular membrane (Köchl et al, ).…”

Functional Analysis of Membrane Proteins Produced by Cell-Free Translation

“…Cell-free system offers all the conveniences required for proper synthesis of MPs. This method offers a high degree of controllability and provides a completely open system allowing direct manipulation of the reaction conditions to optimize protein folding, disulfide bond formation, incorporation of noncanonical amino acids and the synthesis of toxic proteins [4][5][6][7][8][9]. In comparison to conventional cell-based systems, cell-free systems offer rapid protein synthesis, purification and functional analysis.…”

“…This eukaryotic cell-free system offers additional advantages in the form of native, ER-derived endogenous microsomes. Such microsomes contain the entire translocon machinery responsible for proper folding of MPs [8][9][10][11][12][13]. Recently, the potassium channel KcsA was synthesized successfully in this system [13].…”

Qualifying a eukaryotic cell-free system for fluorescence based GPCR analyses

“…The incorporation of biotinylated lipids into vesicles to immobilize GPCR-containing microsomes on streptavidin-coated surfaces was already shown 34 . Demonstrating in genera the feasibility of this approach, cell-free protein synthesis is predestined for high-throughput analysis since protein synthesis reactions can be automatically performed by adding different DNA/mRNA templates to the cell-free reaction in a highly parallel procedure 35 . In addition, the small initial screening volumes in the microliter range can be linearly scaled up to larger volumes for subsequent production processes.…”