Cell Free Protein Expression

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The described conventional in vivo technologies for protein production depend on the cellular integrity and are only suitable for the production of proteins that do not affect the physiology of the host cell.204,205 As already discussed, these methods are limited for the expression of many proteins, e.g., by the formation of inclusion bodies,206,207 by protein instability due to proteolysis,205,208 or by too low yields in case of most membrane proteins. High-level cell-free (CF) expression systems are a promising new tool for the preparative production of difficult proteins (Figure 2). CF systems are principally independent of the cell physiology and they allow direct and immediate control of the reaction at any time. A wide range of critical reaction parameters such as pH, redox potential, and ionic strength can be chosen and adjusted according to the requirements of the specific target protein. Furthermore, any additives that might help to stabilize the recombinant protein after translation can be supplied directly into the reaction and no transport problems through cellular membranes have to be considered. Metabolic conversions or even the breakdown of added substances is usually very low or even not detectable. Options of possible beneficial compounds can include cofactors, ligands, inhibitors, ions, chaperones, and even detergents. The complete and time-independent control in combination with the high flexibility of the reaction conditions provides a challenging opportunity for the preparative production of formerly highly problematic proteins such as membrane proteins, toxins, or unstable proteins.

A unique characteristic of CF expression techniques is the possibility of quickly and easily introducing specific labels into a protein (Figure 2). Labeling of proteins with stable isotopes is indispensable for the structural and functional analysis by NMR techniques and for drug screening and ligand interaction studies. Labeling of proteins with spectrally enhanced amino acids can further be very helpful for the analysis of protein interaction studies.209 The composition and concentration of all low molecular weight substances in the CF reaction is fully defined and the operator has therefore complete control over the amino acid pool of the reaction. Any type of amino acid can thus easily be replaced by a labeled derivative and a 100% label incorporation into the recombinant protein is ensured. The kinetics and

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Figure 2 Schematic illustration of cell-free protein synthesis in a bacterial coupled transcription and translation system. The cell extract contains ribosomes, translation factors acetate kinase, and aminoacyl-tRNA synthetases (ARSs). T7 RNA polymerase and substrates like amino acids, the energy regenerating system components, nucleotide triphosphates (NTPs), tRNAs, and salts are supplied and protein synthesis is initiated by adding template DNA. The incorporation of selected isotopic labeled amino acids (red) can easily be achieved in the cell-free system, leading to a selectively isotopic labeled protein. Regeneration of NTPs is accomplished by an ATP regenerating energy system (yellow) based on the hydrolysis of high-energy substrates in the presence of their cognate kinases. To assist stability and folding of the target proteins, detergents, chaperones, and other supplements may be added to the reaction mixture.

Figure 2 Schematic illustration of cell-free protein synthesis in a bacterial coupled transcription and translation system. The cell extract contains ribosomes, translation factors acetate kinase, and aminoacyl-tRNA synthetases (ARSs). T7 RNA polymerase and substrates like amino acids, the energy regenerating system components, nucleotide triphosphates (NTPs), tRNAs, and salts are supplied and protein synthesis is initiated by adding template DNA. The incorporation of selected isotopic labeled amino acids (red) can easily be achieved in the cell-free system, leading to a selectively isotopic labeled protein. Regeneration of NTPs is accomplished by an ATP regenerating energy system (yellow) based on the hydrolysis of high-energy substrates in the presence of their cognate kinases. To assist stability and folding of the target proteins, detergents, chaperones, and other supplements may be added to the reaction mixture.

efficiency of the production of labeled proteins equals those of the nonlabeled reactions and no laborious optimizations have to be carried out. In contrast to conventional in vivo labeling systems, no switch to auxotrophic host strains and to fermentations in minimal media is necessary. It should be emphasized that there is virtually no background labeling as the target protein is the only protein that is synthesized in substantial amounts during CF synthesis. High-throughput applications attract increasing attention due to the demands of proteomics associated research. CF synthesis also offers a powerful approach as linear DNA templates simply generated by conventional polymerase chain reaction (PCR) can directly be used for the expression of proteins.210 Design of cell-free expression systems

CF expression systems can be set up as pure translation systems with purified mRNA as a template' or alternatively as coupled transcription-translation systems by adding plasmid or linear DNA as a template.211'212 The simplest design of CF expression is a batch mode reaction with one compartment holding a fixed volume of reaction mixture (RM) in a test-tube. While batch systems are easy to set up and highly suitable for high-throughput applications, they are limited by the rapid accumulation of deleterious by-products like free phosphates from nucleotide consumption that apparently form complexes with magnesium ions. In addition, substrates like nucleotide triphosphates (NTPs) and high-energy phosphate donors are rapidly consumed even in the absence of protein synthesis.213 The results are obtained in relatively short reaction times that normally do not exceed 1 h. Recent modifications of the batch system by using a novel NTP regeneration system can help to prevent the accumulation of inorganic phosphate,213 and the supplementation of additional compounds can significantly extend the reaction time and increase the yield of protein synthesis.214-219 Optimized CF batch systems have the potential to reach a high productivity and may become a powerful technique in the future.

Extended reaction times in CF expression can be achieved by using a continuous-flow CF (CFCF) translation device.220 A key feature is the continuous supply of energy and substrates concomitant with the removal of reaction byproducts. The immobilized RM is continuously perfused by a feeding mixture (FM) containing all low molecular weight precursors and substrates and removing deleterious by-products of the protein production. The RM can alternatively be separated from the FM by a membrane with a variable molecular weight cut-off between 10 and 300 kDa.221 However, reduced exchange rates by blocked membranes can cause significant problems. The protein synthesis in a CFCF system can continue for more than 20 h and yields of up to 1 mg recombinant protein permL RM are possible. The CFCF system can either be operated in the translating mode by adding mRNA,222,223 or in the coupled transcription-translation approach by the addition of phage RNA polymerases.221,222,224 Many successfully synthesized proteins like the bacteriophage MS2 coat protein,220 the brome mosaic virus coat protein,220 globin,225 calcitonin,222 DHFR,222,226-228 chloramphenicol acetyltransferase,222,221,229-231 interleukin-2,232 and interleukin-6223 demonstrate the enormous potential of CFCF protein synthesis.

The relatively complex reaction set-up of the continuous flow mode is simplified in continuous-exchange CF (CECF) expression systems.230,233 The RM and FM compartments have fixed volumes and are separated by a dialysis membrane. The simplest device for a CECF set-up is a dialysis bag holding the RM that is placed in a suitable container with the FM,233 but commercially available dialyer device, such as the MicroDialyzer and DispoDialyzer (Spectrum Laboratories, Breda, The Netherlands), can also be used successfully. An additional advantage of the CECF set-up is the accumulation of the synthesized recombinant protein in the RM. High-level synthesis of up to 6 mg recombinant protein permL RM has been reported.230,234 Yields of 1-4mg permL RM for several functionally active proteins like the green fluorescent protein, DHFR, luciferase, and RNA replicase of tobacco mosaic virus have been obtained with eukaryotic wheat germ extracts.235 The CECF system is commercially available as a kit (Roche Diagnostics, Mannheim, Germany) but can also be set up individually.233,236-238 Components of the cell-free reaction

All elements involved in gene expression and protein synthesis have to be added to the RM where transcription and translation takes place (Figure 2). Components like DNA, a highly processive RNA polymerase like the enzyme encoded by the T7 bacteriophage, NTPs, mRNA, tRNA, aminoacyl tRNA synthetases (ARSs), ribosomes, transcription and translation factors as well as amino acids have to be combined in an optimal pH and salt environment. The required high amounts of free energy for the transcription and translation processes are provided by hydrolysis of the triphosphates ATP and GTP. Crucial for each CF system is therefore an efficient ATP regenerating energy system in order to maintain the NTP concentrations over a long period of time. Conventional energy systems are based on high-energy phosphate donors such as phosphoenol pyruvate in combination with pyruvate kinase,211,239 creatine phosphate and creatine kinase,234 or acetyl phosphate with acetate kinase.240 Preparation of cell-free extracts

The quality of the cell extract is crucial for the success of a CF system. The extract represents usually a crude cell lysate which contains most of the essential high molecular weight components for translation. Only T7 RNA polymerase and certain enzymes for the energy regeneration have to be supplied. While many organisms could potentially serve as an extract source, lysates based on the cell types of E. coli, wheat germ, and rabbit reticulocytes have been well established.

The bacterial source of choice for the preparation of CF extracts are RNAse-deficient E. coli strains, e.g., A19236 or BL21.237'241 Extracts of E. coli S30 represent the soluble fraction after centrifugation of crude lysates at 30000g. Endogenous mRNA is removed during a preincubation step of the cell extract either with high salt236 or with added nucleotides and amino acids. This 'runoff' step releases endogenous mRNA from the ribosomes that will then subsequently become destroyed by endogenous ribonucleases.242 Alternatively, isolated ribosomes can be added to an S100 extract, centrifuged at 100000g.243,244 Extracts of E. coli are used in coupled transcription-translation CF systems due to the favorable use of T7 RNA polymerase. Extracts of E. coli work well in a wide temperature range between 24 and 38 °C with an optimum at 37 °C. Due to the relatively simple extract preparation procedure combined with high productivity, the E. coli CF system is the most commonly used in vitro protein expression technique and up to 6 mg of protein per ml RM can be synthesized.234

A well-defined system using 31 individually purified enzymes isolated with conventional expression systems together with purified ribosomes is termed the 'protein synthesis using recombinant elements' (PURE) system.246The advantage of the PURE system is the absence of any inhibitory substances such as nucleases, proteases, and enzymes that hydrolyze nucleoside triphosphates.

The most convenient and promising eukaryotic CF translation system is based on wheat germs isolated from dry wheat seeds.247 Recent modifications resulted in extracts with a high degree of stability and activity.235 Important for an enhanced translation efficiency in the wheat germ CF system are the 5' and 3' UTRs of eukaryotic mRNAs that play a crucial role in the regulation of gene expression by controlling mRNA translational efficiency, stability, and localization. An optimal 5' UTR that should therefore be added to mRNAs is the so-called omega sequence (O71) of tobacco mosaic virus. For the 3' UTR the length is more important for an efficient translation than the sequence.248 Wheat germ extracts possess only low levels of endogenously expressed mRNAs and therefore can be directly used for the expression of templates.249 The optimal reaction temperature is in the range of 20-27 °C, but can be increased to up to 32 °C for higher expression of some templates.252 The reaction continues up to 60 h and amounts of 1-4 mg of recombinant protein per mL RM can be obtained.235

Lysates of rabbit reticulocytes are obtained from blood cells of anemic rabbits that provide a high number of reticulocytes or proerythrocytes. Endogenous mRNA is removed by treatment with micrococcal Ca2 + -dependent RNase.253 The yields of recombinant protein can also be in the range of mgpermL RM, whereas the expression yields of the wheat germ system are usually higher.254 This system works in an optimal temperature range of 30-38 °C.255

In principle, the choice of extract source, either prokaryotic or eukaryotic, for CF synthesis should be chosen according to the origin and biochemical nature of the protein of interest. In general E. coli-based systems gain in terms of their higher translation rates, better compatibility with combined transcription-translation formats, easier preparation of extracts as well as reaction set-up, and the availability of mutants with reduced degradative activities. On the other hand they suffer from high degradation of genetic messages, shorter lifetimes, and a great tendency of protein aggregation. Eukaryotic extracts are mainly limited by lower translation rates and the complexity of the genetic constructs that are required for an effective expression. Positive aspects of eukaryotic CF systems are their higher stability and longer lifetime in addition to a better compatibility with eukaryotic mRNAs and the synthesis of eukaryotic proteins.

CECF expression kits based on E. coli and wheat germ extracts are commercially available (Roche Diagnostics, Mannheim, Germany). The successful production of more than 40 proteins of different origins, including various enzymes, receptors, hormones, antibodies, and regulatory proteins has been shown by various laboratories.256,257 Other commercial systems like Expressway (Invitrogen, Carlsbad, CA) focus on the batch mode with expression amounts of up to 1 mgmL _ 1. Cell-free synthesis of membrane proteins

Membrane proteins today represent less than 1% of the available three-dimensional protein structures, although this is in contrast to their immense medical importance as an estimate of 60-70% of current drug targets are based on membrane proteins. The key bottleneck has been the lack of reliable technologies that ensure the production of a broad variety of recombinant membrane proteins in the required amounts.258 Toxicity to the host cell, protein aggregation, and miss-folding of overproduced membrane proteins very often result in low yields. Furthermore, the overexpression of integral membrane proteins often causes cell death by overloading the cytoplasmic membranes or by disrupting the membrane integrity. Two protein groups of outstanding pharmaceutical relevance are multidrug resistance transporters of bacterial pathogens and GPCRs as the basic elements of the eukaryotic signal transduction machinery. Preparative-scale expression of GPCRs has not been obtained in most cases, and it is always subject of tedious and long-lasting optimizations.194,195,259 A promising perspective is that the newly developed CF translation systems offer a powerful alternative for overcoming the tremendous expression barriers for membrane proteins (Figure 1). It has recently been shown that functionally active integral membrane proteins, especially small multidrug transporters, GPCR proteins, a light-harvesting membrane protein, and ion channels were expressed in high yields of mg amounts per mL RM in an E. coli-based CECF system.236,238,260-262 The proteins could also be functionally reconstituted into proteoliposomes and isotopically labeled for NMR investigations.236 Even the addition of mild detergents does not interfere with the translation activity of the CECF systems and results directly in the soluble and functional expression of several integral membrane proteins.238,260,261 The combination of isotopic labeling and membrane protein expression demonstrates the high potential of the CF method for functional and structural membrane protein research. Finally it should be mentioned that the preparation of membrane protein samples ready for a structural analysis by, e.g., NMR techniques is possible in less than 2 days by using CF expression systems and the structural characterization of even very difficult protein families like the GPCR proteins becomes now feasible. Cell-free synthesis of disulfide-bridged proteins

Disulfide-bonded proteins are rarely expressed in traditional expression systems due to their requirements for oxidizing conditions which in eukaryotes are only found in the lumen of the endoplasmatic reticulum (or in the periplasm of prokaryotes). The open character of CF systems allows the direct addition of purified chaperones like the already discussed eukaryotic protein disulfide isomerase (PDI) or bacterial Dsb derivatives with success of the functional expression of single chain antibodies.263 CF expression reactions are usually operated in the presence of reducing agents like dithiothreitol that stabilize the protein transcription-translation machinery but provide less favorable conditions for the synthesis of disulfide-bonded proteins. However, the use of a dithiothreitol-deficient wheat germ extract in the presence of the PDI chaperone efficiently synthesizes a single-chain antibody variable fragment with dual disulfide bonds.264 More recently, the problem of the CF expression of disulfide-bonded proteins was overcome by using a combination of iodoacetamide-treated extract, a suitable glutathione redox buffer, and the addition of disulfide bond shuffling chaperones like Skp and DsbC.265 A recombinant plasminogen activator protein with nine disulfide bonds could productively be expressed with this approach.

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