BMS Interview: Dr. Stefan Kubick
1. What is a cell-free protein expression system?
In the 1960s, Marshall Nirenberg and Heinrich Matthaei pioneered the use of prokaryotic enzyme extracts to characterize a range of essential factors in protein synthesis. One result of their famous “PolyU” experiment was to demonstrate the role of a ‘template RNA’ in protein synthesis – a milestone on the road to the ultimate discovery of the base triplet as the coding unit for amino acids.
At the end of the 1980s, it was possible to significantly boost the capabilities of cell lysates, thanks especially to the work of Alexander Spirin, finally enabling the synthesis of larger quantities of proteins in cell-free systems. The “Continuous-Exchange Cell-Free” process patented by Spirin was based on a small bioreactor with a synthesis chamber, coupled to a reservoir tank that, via a dialysis membrane, continuously supplied the reaction with fresh consumables such as ATP and GTP as sources of energy, plus all of the necessary amino acids as building blocks for the protein synthesis itself. Small-molecule inhibitors were also removed from the synthesis chamber, as their increasing concentration disrupted the process. Left in the reaction chamber were the coding nucleic acids and the ribosomal machinery for protein synthesis, plus the gradually accumulating, newly synthesized proteins. This technology was the first to offer continuous cell-free synthesis of proteins over a period of up to 24 hours, which represented a significant improvement in protein output compared to other systems.
Cell-free protein synthesis systems, also termed in vitro translation systems, expand the diversity of protein expression in a remarkable way. The intense development and analysis of pro- and eukaryotic in vitro translation systems are permanently growing fields of research. Open cell-free systems are designed for the synthesis of complex membrane proteins, glycoproteins and cytosolic proteins with the addition of posttranslational modifications onto the growing nascent polypeptide chain. The development of translationaly active cell lysates into highly productive systems, the generation and optimization of particular DNA and RNA templates for different cell-free systems and the optimization of reaction conditions in newly developed in vitro translation systems are part of our current research investigations.
2. What is the yield of expressed proteins?
Cell-free systems based on E. coli lysates utilise batch based and dialysis based procedures to deliver protein yields up to 5 mg/ml. Improvements here have focused primarily on ensuring high protein content in a soluble and functional form. In contrast to the conventional expression of proteins in cells and bacteria, the open, cell-free systems permit the configuration of the ionic milieu, the reaction temperature and the dosing of mild detergents, as well as folding helpers (chaperones) even during protein synthesis. Those proteins, termed “difficult-to-express proteins”, which to date have not been successfully expressed or which exhibit cytotoxic effects and kill off the host cell, can now be readily expressed by deploying an efficient template with a protein-specific supplementation of the translation reaction in automated, highly parallel cell-free systems.
The expression of functionally active eukaryotic proteins requires not only the coordinated interaction of the protein expression machinery but also the integrity of all co- and post-translationally protein modifying components. If human genes are to be manufactured in a modified and simultaneously active form, this is best done in a system that most closely corresponds to the natural environment of these proteins – and at the point in time of synthesis. For this reason, there is large-scale use of cultivated mammalian cells – and human cell lines in particular – for the human-identical manufacturing of pharmacologically relevant proteins in eukaryotic cell-free systems. The decisive advantages of cell-free protein synthesis in eukaryotic in vitro translation systems, such as the high-throughput synthesis of continuously active, cytotoxic membrane proteins, can be combined with biochip-based, impedimetric and electrophysiological functional analysis. A typical yield for a protein synthesized in a eukaryotic cell-free system is 10 to 100 µg per ml depending on the individual protein to be produced.
3. How do you perform glycosylation in a cell-free system?
Cultivated eukaryotic cell lines possess the necessary subcellular structures to perform protein glycosylation and the enzymatic activities found within them are a prerequisite to perform these protein modifications. They therefore constitute an ideal starting substrate for the manufacture of eukaryotic in vitro translation systems. In this way, the general advantages of cell-free protein synthesis– such as the possibility of synthesising cytotoxic and labelled proteins – are combined with a rapid and easy to manage expression procedure for functionally active eukaryotic proteins. Due to the procedure utilised for the homogenisation of eukaryotic cells, intracellular, vesicular structures are retained in a functionally active form in the lysate. These microsomal elements are deployed for co-translational translocation and the immobilisation of cell-free synthesised membrane proteins on chip surfaces. The functional integrity of these subcellular components can be demonstrated by means of signal peptide cleavage, as well as the glycosylation of cell-free synthesised proteins. This enormous potential of eukaryotic in vitro translation systems is also utilised in the manufacture of antibody fragments, which involves the accumulation of defined,posttranslationally modified and fluorescence- labelled antibody fragments in proteoliposomes.
4. How do you perform quality checking?
Many issues, such as lysate preparation, optimization of reaction conditions, and the design of the expression vector are taken into consideration for cell-free synthesis of functional proteins. Particularly, the latter is a crucial aspect for high-yield in vitro protein production. The performance of the cell-free systems is evaluated by synthesizing functional cytosolic proteins, posttranslationally modified proteins and membrane-spanning proteins. To monitor protein quality and quantity, reaction mixtures are supplemented with 14C labeled leucine. De novo synthesized proteins are routinely analyzed by autoradiography and radioactively labeled proteins are visualized using a phosphorimager system. Protein yield is determined by hot TCA precipitation. Additionally, detailed analysis is performed by mass spectrometry. Finally, cell-free produced ion channels for example, are analyzed by electrophysiological measurements.
5. Do you believe that one can rely on the system to perform complex eukaryotic glycosylations?
Various cell-free protein synthesis systems based on cultured insect cells as well as cultured mammalian cells have been developed in the past decade in order to address the synthesis of posttranslationally modified proteins.
The use of recently established lysates derived from these cultured insect cells and mammalian cells is preferred, as these extracts already contain endogenous microsomes derived from the endoplasmic reticulum of the cultured cells and these cell-free protein synthesis platforms have the potential to perform signal sequence processing and N-linked glycosylation. An emerging number of publications illustrates the application of these cell-free systems for the synthesis of co- and post-translationally modified proteins. Additionally, target proteins could be engineered in a desired manner by making use of the accessible nature of the cell-free protein synthesis reaction. In this context, bioorthogonal systems provide a promising tool for the incorporation of chemoselective reactive amino acids into synthesized proteins at defined positions by expanding the genetic code. These amino acids can subsequently be modified in a desired manner by the addition of a corresponding reaction partner, e.g. chemically synthesized glyco polymers, resulting in novel characteristics of the target protein. Several bioorthogonal and chemoselective reactions have been identified and applied in cell-free systems in recent years and this technology will obviously expand the number of applications of cell-free protein synthesis in the field of pharmaceutical research and development.
6. Do you see the cell-free expression system more as a vehicle of basic research or as a tool for industry?
High-throughput protein expression platforms are becoming increasingly important. A unique and exciting development is the potential to enable the production of personalized medicines. Cell-free systems in particular, have many advantages for meeting this need. Cell-free expression methodology is well suited for pharmaceutical protein expression and engineering and will probably become more commonly used in the future. In the past decade, substantial progress has been made in the development of novel mammalian-based cell-free protein synthesis systems. However, there are still limitations which are currently in the focus of research and developmental efforts. Ongoing challenges in the field of mammalian-based cell-free protein synthesis are the optimization of already established platforms and the development of novel systems in order to enable even higher protein yields and lower manufacturing costs while facilitating the synthesis of a huge number of biologically active target proteins. By applying a combination of cell-free protein synthesis, high-throughput screening technologies and protein engineering, the chances of obtaining a therapeutic protein with optimal properties such as efficacy, stability and solubility, can only be increased. In this way, cell-free protein synthesis will be a true alternative to cell based protein expression systems currently used in industrial applications.
7. Is synthesis of functional biomarkers possible?
‘‘Lab-on-chip“ technologies are believed to be one of the mainstream technologies within the next centuries and high-throughput multiplex immunoassays that measure hundreds of proteins in complex biological matrices in parallel have become significant tools for quantitative proteomics studies, diagnostic discovery, and biomarker-assisted drug development. In this context cell-free protein synthesis is an essential prerequisite for the „just-in-time on-chip synthesis“ of biomarkers and the development of label-free assays based on these freshly synthesized active proteins.
8. Where do you see the highest potential for future application?
While used for decades as a foundational research tool for understanding transcription and translation, recent advances have made possible cost-effective microscale to manufacturing scale cell-free synthesis of complex proteins. Protein yields exceed grams protein produced per liter reaction volume, batch reactions last for multiple hours, costs have been reduced orders of magnitude and reaction scale has reached the 100-liter milestone. Recent advances have inspired new applications in the synthesis of protein libraries for functional genomics, the production of personalized medicines, and the expression of tailor-made biological recognition elements including membrane-bound enzymes and receptors.
Photo and text: Dr. Dana Kikic