Cell-free Synthesis of Membrane Proteins
New Production Pipelines for Tricky Molecules
- Fig. 1: Cellular versus cell-free membrane protein production. Key benefits of cell-free systems are: (I) Open access enabling addition of stabilizers; (II) Solubilization of membrane proteins directly after translation without transport requirements; (III) Elimination of toxic effects; (IV) No background expression.
- Fig. 2: Key options to modulate expression efficiencies and sample quality
- Fig. 3: Cell-free expression protocol development in 96 well formats. Automated screening of compound concentrations and additive affects results in optimized expression conditions.
- Fig. 4: Cell-free expression modes
- Friederike Junge, Daniel Schwarz, Prof. Dr. Volker Dötsch and Dr. Frank Bernhard from the Goethe University Frankfurt am Main
Sample preparation for the molecular analysis of membrane proteins is a highly challenging task. Implementing conventional living-cell based production machineries are frequently not appropriate as increased copy numbers of membrane proteins can have a variety of toxic side effects on essential physiological processes of the host cells. A new approach avoids living hosts by using cell extracts and has thus emerged as powerful technology for the rapid and high-level production of these difficult targets.
Challenges of Membrane Protein Production
Membrane proteins have diverse functions within a cell, such as transporters, receptors, channels or enzymes and comprise approximately 30% of averaged cellular proteomes. Any traffic, uptake or excretion of substances, communication of cells with their environment as well as signal perception is strictly controlled by membrane proteins. These central roles in any living organism brings them into focus as one of the most prominent targets of pharmaceutical industries. Despite their eminent importance, the majority of membrane proteins still remain as white spots on the landscape of protein characterization and knowledge of their molecular structures, a basic requirement for directed drug design, thus lacking far behind if compared to that of soluble globular proteins. Their intrinsic hydrophobic nature in combination with complex biosynthesis pathways, requiring sophisticated helper networks for the efficient targeting and insertion into destination membranes, often does not match with established cellular production systems. A variety of toxic effects e.g. blocking the limited membrane space or affecting sensitive concentration gradients can additionally result in poor growth or even death of producer cells. Low yields or insufficient sample quality are therefore frequent consequences of membrane protein production in living cells, regardless whether bacteria, yeasts or higher eukaryotic cells are used (fig. 1).
Cell-free Expression Technologies
Avoiding living organisms by cell-free expression offers a completely new principle of membrane protein production and virtually eliminates most central bottlenecks of conventional approaches, such as toxicity, targeting or translocation (fig.
1). Cell extracts are prepared by disrupting vigorously growing cells and harvesting ribosome enriched fractions, thus keeping the essential translation machinery intact while most other physiological pathways are disrupted. Bacteria as well as eukaryotic cells from wheat germs, rabbit reticulocytes or protozoa are currently used as major extract sources. Elimination of endogenous mRNAs allows the background free production of target proteins in subsequent reactions. Cell-free expression can be operated as two compartment continuous exchange reaction, where low molecular weight compounds like amino acids and energy precursors are replenished and potentially inhibitory by-products are continuously removed (fig. 1). Reduction of complexity in membrane protein production down to the basic translation process results in high success rates combined with considerable efficiencies. Hundreds of even very difficult membrane proteins including large eukaryotic transporters, aquaporins or G-protein coupled receptors have already been successfully produced by this technique. Production efficiencies can be several milligrams of target protein in a single milliliter of reaction. Reaction volumes can therefore be kept small and are usually in the range of only few milliliters. Fascinating as well is the speediness of the reaction completing the whole expression process within less than 24 hours.
Modulating Sample Quality
A valuable feature of cell-free expression is its open accessible nature as the cellular integrity is destroyed and the reaction is no longer enclosed by impermeable membrane barriers. This characteristic allows the supplementation of potentially beneficial compounds into the reaction at any time point of the expression process. Stabilizing substances like substrates, inhibitors, cofactors or chaperones could support the functional folding of synthesized membrane proteins. Oxidizing conditions can be manipulated in order to direct disulfide bridge formations. Modulating the expression environment becomes therefore a major and powerful tool for sample quality optimization of expressed proteins. This option is most prominent by cell-free expression, while only very limited available in cellular expression systems (fig. 2). Type and concentration of additives can play pivotal roles and robotic screening programs have been established in order to generate individualized expression protocols for optimal sample production (fig. 3).
Cell-free Expression Modes
Due to their hydrophobic nature, freshly translated membrane proteins will instantly precipitate in the aqueous reaction environment (fig. 4). This P-CF (precipitate forming) mode resembles the inclusion body formation known from expression in bacterial cells. However, at least partly folded structures seem to remain, as several even complex membrane proteins can be obtained from these precipitates in functional form by resolubilization in relatively mild detergents. In the D-CF (detergent based) expression mode, detergents are added into the reaction and membrane proteins are soluble expressed by direct insertion into micelles. This mode is unique to cell-free systems and the translation machinery in the cell extracts tolerates a wide variety of detergents even in high concentrations. The purified detergent solubilized membrane proteins could directly be analyzed by structural approaches like Nuclear Magnetic Resonance or X-ray crystallography. In the L-CF (lipid based) expression mode, lipids can be added into the reaction as microsomes, defined preformed liposomes or planar membrane discs in form of nanodiscs. Also providing combinations of detergents and lipids as bicelles or lipo-micelles can considerably trigger the stability of inserted membrane proteins.
Conclusions and Perspectives
Cell-free expression is still a relatively new approach and further system developments are continuously published. Lists of solubilizing compounds used in the D-CF and L-CF modes are rapidly expanding and the variety of extract sources will also further increase. But already today cell-free expression is the most versatile tool to produce membrane proteins, giving nearly unlimited options to support stability and folding as soon as the target proteins come out of the ribosome. In particular structural approaches benefit from cell-free expression as sample production is fast and cost effective. Nuclear Magnetic Resonance profits from the fast and easy generation of protein samples labeled with stable isotopes. Uniform or combinatorial labeling of cell-free expressed proteins with amino acid derivatives is highly efficient and opens completely new avenues for the structural analysis of membrane proteins.
References are available from the authors.