Marlitt Stech

Proteins are the main source of drug targets and some of them possess therapeutic potential themselves. Among them, membrane proteins constitute approximately 50% of the major drug targets. In the drug discovery pipeline, rapid methods for producing different classes of proteins in a simple manner with high quality are important for structural and functional analysis. Cell-free systems are emerging as an attractive alternative for the production of proteins due to their flexible nature without any cell membrane constraints. In a bioproduction context, open systems based on cell lysates derived from different sources, and with batch-to-batch consistency, have acted as a catalyst for cell-free synthesis of target proteins. Most importantly, proteins can be processed for downstream applications like purification and functional analysis without the necessity of transfection, selection, and expansion of clones. In the last 5 years, there has been an increased availability of new cell-free lysates derived from multiple organisms, and their use for the synthesis of a diverse range of proteins. Despite this progress, major challenges still exist in terms of scalability, cost effectiveness, protein folding, and functionality. In this review, we present an overview of different cell-free systems derived from diverse sources and their application in the production of a wide spectrum of proteins. Further, this article discusses some recent progress in cell-free systems derived from Chinese hamster ovary and Sf21 lysates containing endogenous translocationally active microsomes for the synthesis of membrane proteins. We particularly highlight the usage of internal ribosomal entry site sequences for more efficient protein production, and also the significance of site-specific incorporation of non-canonical amino acids for labeling applications and creation of antibody drug conjugates using cell-free systems. We also discuss strategies to overcome the major challenges involved in commercializing cell-free platforms from a laboratory level for future drug development.

Cell-free protein synthesis (CFPS) represents a promising technology for efficient protein production targeting especially so called "difficult-to-express" proteins whose synthesis is challenging in conventional in vivo protein production platforms. Chinese hamster ovary (CHO) cells are one of the most prominent and safety approved cell lines for industrial protein production. In this study we demonstrated the ability to produce high yields of various protein types including membrane proteins and single chain variable fragments (scFv) in a continuous exchange cell-free (CECF) system based on CHO cell lysate that contains endogenous microsomal structures. We showed significant improvement of protein yield compared to batch formatted reactions and proved biological activity of synthesized proteins using various analysis technologies. Optimized CECF reaction conditions led to membrane protein yields up to 980 µg/ml, which is the highest protein yield reached in a microsome containing eukaryotic cell-free system presented so far.

Internal ribosome entry site (IRES) elements found in the 59 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 cellfree 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.

Antibodies are indispensable tools for basic research as well as diagnostic and therapeutic applications. Consequently, the development of alternative manufacturing strategies which circumvent the hurdles connected to conventional antibody production technologies is of enormous interest. To address this issue, we demonstrate the synthesis of complex antibody formats, in particular immunoglobulin G (IgG) and single-chain variable fragment Fc fusion (scFv-Fc), in a microsome-containing cell-free system based on translationally active chinese hamster ovary (CHO) cell lysates. To mimic the environment for antibody folding and assembly present in living cells, antibody genes were fused to an endoplasmic reticulum (ER)-specific signal sequence. Signal-peptide induced translocation of antibody polypeptide chains into the lumen of ER microsomes was found to be the prerequisite for antibody chain assembly and functionality. In this context, we show the rapid synthesis of antibody molecules in different reaction formats, including batch and continuous-exchange cell-free (CECF) reactions, depending on the amount of protein needed for further analysis. In addition, we demonstrate site-specific and residue-specific labeling of antibodies with fluorescent non-canonical amino acids. In summary, our study describes a novel antibody production platform which combines the highly efficient mammalian protein folding machinery of CHO cells with the benefits of cell-free protein synthesis.

Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG) molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv) and antigen binding fragments (Fab), have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufacture highly specific molecular recognition tools. However, as these technologies are accompanied by the drawbacks of being rather time-consuming and cost-intensive, efficient and powerful cell-free protein synthesis systems have been developed over the last decade as alternatives. So far, prokaryotic cell-free systems have been the focus of interest. Recently, eukaryotic in vitro translation systems have enriched the antibody production pipeline, as these systems are able to mimic the natural pathway of antibody synthesis in eukaryotic cells. This review aims to overview and summarize the advances made in the production of antibodies and antibody fragments in cell-free systems.