From its start as a small‐scale in vitro system to study fundamental translation processes, cell‐free protein synthesis quickly rose to become a potent platform for the high‐yield production of proteins. In contrast to classical in vivo protein expression, cell‐free systems do not need time‐consuming cloning steps, and the open nature provides easy manipulation of reaction conditions as well as high‐throughput potential. Especially for the synthesis of difficult to express proteins, such as toxic and transmembrane proteins, cell‐free systems are of enormous interest. The modification of the genetic code to incorporate non‐canonical amino acids into the target protein in particular provides enormous potential in biotechnology and pharmaceutical research and is in the focus of many cell‐free projects. Many sophisticated cell‐free systems for manifold applications have been established. This review describes the recent advances in cell‐free protein synthesis and details the expanding applications in this field.

Protein expression systems are widely used in biotechnology and medicine for the efficient and economic production of therapeutic proteins. Today, cultivated Chinese hamster ovary (CHO) cells are the market dominating mammalian cell-line for the production of complex therapeutic proteins. Despite this outstanding potential of CHO cells, no high-yield cell-free system based on translationally active lysates from these cells has been reported so far. To date, CHO cell extracts have only been used as a foundational research tool for understanding mRNA translation (Lodish et al., 1974;McDowell et al., 1972). In the present study, we address this fact by establishing a novel cell-free protein expression system based on extracts from cultured CHO cells. Lysate preparation, adaptation of in vitro reaction conditions and the construction of particular expression vectors are considered for high-yield protein production. A specific in vitro expression vector, which includes an internal ribosome entry site (IRES) from the intergenic region (IGR) of the Cricket paralysis virus (CrPV), has been constructed in order to obtain optimal performance. The IGR IRES is supposed to bind directly to the eukaryotic 40S ribosomal subunit thereby bypassing the process of translation initiation, which is often a major bottleneck in cell-free systems. The combination of expression vector and optimized CHO cell extracts enables the production of approximately 50 mg/mL active firefly luciferase within 4 h. The batch-type cell-free coupled transcription-translation system has the potential to perform post-translational modifications, as shown by the glycosylation of erythropoietin. Accordingly, the system contains translocationally active endogenous microsomes, enabling the co-translational incorporation of membrane proteins into biological membranes. Hence, the presented in vitro translation system is a powerful tool for the fast and convenient optimization of expression constructs, the specific labeling of integral membrane proteins and the cell-free production of posttranslationally modified proteins.

Myriads of odorous molecules that vary widely in structure are nevertheless readily detected and discriminated by the sense of smell, but how this is achieved by the olfactory system has been a long-standing puzzle. Several different models have been proposed, and previous observations indicate that the recognition sites for odorous molecules could be G-protein-coupled receptor proteins, an idea supported by the discovery of a new gene family that probably encodes a diversity of odorant receptors. Here we report the identification of new members of the gene family encoding putative odorant receptors and demonstrate that they are indeed transcribed in olfactory receptor neurons. Furthermore, the receptor-encoding complementary DNA is expressed in non-neuronal surrogate cells, which generate second messenger responses upon stimulation with appropriate odorants, indicating that the receptors recognize odorants and couple to G proteins of the host cells.

Internal ribosome entry site (IRES) elements found in the 5′ untranslated region of mRNAs enable translation initiation in a cap-independent manner, thereby representing an alternative to cap-dependent translation in cell-free protein expression systems. However, IRES function is largely species-dependent so their utility in cell-free systems from different species is rather limited. A promising approach to overcome these limitations would be the use of IRESs that are able to recruit components of the translation initiation apparatus from diverse origins. Here, we present a solution to this technical problem and describe the ability of a number of viral IRESs to direct efficient protein expression in different eukaryotic cell-free expression systems. The IRES from the intergenic region (IGR) of the Cricket paralysis virus (CrPV) genome was shown to function efficiently in four different cell-free systems based on lysates derived from cultured Sf21, CHO and K562 cells as well as wheat germ. Our results suggest that the CrPV IGR IRES-based expression vector is universally applicable for a broad range of eukaryotic cell lysates. Sf21, CHO and K562 cell-free expression systems are particularly promising platforms for the production of glycoproteins and membrane proteins since they contain endogenous microsomes that facilitate the incorporation of membrane-spanning proteins and the formation of post-translational modifications. We demonstrate the use of the CrPV IGR IRES-based expression vector for the enhanced synthesis of various target proteins including the glycoprotein erythropoietin and the membrane proteins heparin-binding EGF-like growth factor receptor as well as epidermal growth factor receptor in the above mentioned eukaryotic cell-free systems. CrPV IGR IRES-mediated translation will facilitate the development of novel eukaryotic cell-free expression platforms as well as the high-yield synthesis of desired proteins in already established systems.

a b s t r a c tWhen taking up the gauntlet of studying membrane protein functionality, scientists are provided with a plethora of advantages, which can be exploited for the synthesis of these difficult-to-express proteins by utilizing cell-free protein synthesis systems. Due to their hydrophobicity, membrane proteins have exceptional demands regarding their environment to ensure correct functionality. Thus, the challenge is to find the appropriate hydrophobic support that facilitates proper membrane protein folding. So far, various modes of membrane protein synthesis have been presented. Here, we summarize current state-of-the-art methodologies of membrane protein synthesis in biomimeticsupported systems. The correct folding and functionality of membrane proteins depend in many cases on their integration into a lipid bilayer and subsequent posttranslational modification. We highlight cell-free systems utilizing the advantages of biological membranes.