Eukaryotic Cell-free Protein Synthesis Research Articles

Solubilization of Oligomeric Cell-Free Synthesized Proteins Using SMA Copolymers.

Although membrane proteins are abundant in nature, their investigation is limited due to bottlenecks in heterologous overexpression and consequently restricted accessibility for downstream applications. In this chapter, we address these challenges by presenting a fast and straightforward synthesis platform based on eukaryotic cell-free protein synthesis (CFPS) and an efficient solubilization strategy using styrene-maleic acid (SMA) copolymers. We demonstrate CFPS of TWIK-1, a dimeric ion channel, based on Sf21 (Spodoptera frugiperda) insect lysate showing homooligomerization and N-glycosylation enabled by endoplasmic reticulum-derived microsomes. Furthermore, we employ SMA copolymers for protein solubilization, which preservesthe native-like microsomal environment. This approach not only retains the solubilized protein's suitability for downstream applications but also maintains the oligomerization and glycosylation of TWIK-1 post-solubilization. We validate the solubilization procedure using autoradiography, particle size analysis, and biomolecular fluorescence assay and confirm the very efficient, structurally intact solubilization of cell-free synthesized TWIK-1.

Enhancing the performance of a mutant pyrrolysyl-tRNA synthetase to create a highly versatile eukaryotic cell-free protein synthesis tool

Modification of proteins with a broad range of chemical functionalities enables the investigation of protein structure and activity by manipulating polypeptides at single amino acid resolution. Indeed, various functional groups including bulky non-canonical amino acids like strained cyclooctenes could be introduced by the unique features of the binding pocket of the double mutant pyrrolysyl-tRNA synthetase (Y306A, Y384F), but the instable nature of the enzyme limits its application in vivo. Here, we constructed a cell-free protein production system, which increased the overall enzyme stability by combining different reaction compartments. Moreover, a co-expression approach in a one-pot reaction allowed straightforward site-specific fluorescent labeling of the functional complex membrane protein cystic fibrosis transmembrane conductance regulator. Our work provides a versatile platform for introducing various non-canonical amino acids into difficult-to-express proteins for structural and fluorescence based investigation of proteins activity.

Scaling eukaryotic cell-free protein synthesis achieved with the versatile and high-yielding tobacco BY-2 cell lysate.

Eukaryotic cell-free protein synthesis (CFPS) can accelerate expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and difficulties scaling such systems have prevented their widespread adoption in protein research and manufacturing. Here, we provide detailed demonstrations for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in 48 h, complete with native disulfide bonds and N-glycosylation. An optimized version of the technology is commercialized as ALiCE® and advances in scaling of BYL production methodologies now allow scaling of eukaryotic CFPS reactions. We show linear, lossless scale-up of batch mode protein expression from 100 µL microtiter plates to 10 and 100 mL volumes in Erlenmeyer flasks, culminating in preliminary data from a litre-scale reaction in a rocking-type bioreactor. Together, scaling across a 20,000x range is achieved without impacting product yields. Production of multimeric virus-like particles from the BYL cytosolic fraction were then shown, followed by functional expression of multiple classes of complex, difficult-to-express proteins using the native microsomes of the BYL CFPS. Specifically: a dimeric enzyme; a monoclonal antibody; the SARS-CoV-2 receptor-binding domain; a human growth factor; and a G protein-coupled receptor membrane protein. Functional binding and activity are demonstrated, together with in-depth PTMcharacterization of purified proteins through disulfide bond and N-glycan analysis. Taken together, BYL is a promising end-to-end R&D to manufacturing platform with the potential to significantly reduce the time-to-market for high value proteins and biologics.

One to one comparison of cell-free synthesized erythropoietin conjugates modified with linear polyglycerol and polyethylene glycol

With more than 20 Food and Drug Administration (FDA)-approved poly (ethylene glycol) (PEG) modified drugs on the market, PEG is the gold standard polymer in bioconjugation. The coupling improves stability, efficiency and can prolong blood circulation time of therapeutic proteins. Even though PEGylation is described as non-toxic and non-immunogenic, reports accumulate with data showing allergic reactions to PEG. Since PEG is not only applied in therapeutics, but can also be found in foods and cosmetics, anti-PEG-antibodies can occur even without a medical treatment. Hypersensitivity to PEG thereby can lead to a reduced drug efficiency, fast blood clearance and in rare cases anaphylactic reactions. Therefore, finding alternatives for PEG is crucial. In this study, we present linear polyglycerol (LPG) for bioconjugation as an alternative polymer to PEG. We report the conjugation of LPG and PEG by click-chemistry to the glycoprotein erythropoietin (EPO), synthesized in a eukaryotic cell-free protein synthesis system. Furthermore, the influence of the polymers on EPOs stability and activity on a growth hormone dependent cell-line was evaluated. The similar characteristics of both bioconjugates show that LPGylation can be a promising alternative to PEGylation.

Targeted esterase-induced dye (TED) loading supports direct calcium imaging in eukaryotic cell-free systems.

Calcium imaging is an important functional tool for analysing ion channels, transporters and pumps for drug screening in living cells. Depicted eukaryotic cell-free systems utilize microsomes, derived from the endoplasmic reticulum to incorporate the synthesized membrane proteins-like ion channels. Carboxylesterase is required to cleave the acetoxymethyl ester moiety of the chemical calcium indicators in order to ensure its immobility across the endoplasmic reticulum membrane. Absence or an inadequate amount of carboxylesterase in the endoplasmic reticulum of different eukaryotic cells poses a hindrance to perform calcium imaging in microsomes. In this work, we try to overcome this drawback and adapt the cell-based calcium imaging principle to a cell-free protein synthesis platform. Carboxylesterase synthesized in a Spodoptera frugiperda Sf21 lysate translation system is established as a viable calcium imaging tool in microsomes. Cell-free synthesized carboxylesterase inside microsomes is validated with esterase and dye loading assays. Native proteins from the endoplasmic reticulum, such as ryanodine channels and calcium ATPase, are analysed. Cell-free synthesized transient receptor potential channels are used as model proteins to demonstrate the realization of this concept.

Accelerating the Production of Druggable Targets: Eukaryotic Cell-Free Systems Come into Focus

In the biopharmaceutical pipeline, protein expression systems are of high importance not only for the production of biotherapeutics but also for the discovery of novel drugs. The vast majority of drug targets are proteins, which need to be characterized and validated prior to the screening of potential hit components and molecules. A broad range of protein expression systems is currently available, mostly based on cellular organisms of prokaryotic and eukaryotic origin. Prokaryotic cell-free systems are often the system of choice for drug target protein production due to the simple generation of expression hosts and low cost of preparation. Limitations in the production of complex mammalian proteins appear due to inefficient protein folding and posttranslational modifications. Alternative protein production systems, so-called eukaryotic cell-free protein synthesis systems based on eukaryotic cell-lysates, close the gap between a fast protein generation system and a high quality of complex mammalian proteins. In this study, we show the production of druggable target proteins in eukaryotic cell-free systems. Functional characterization studies demonstrate the bioactivity of the proteins and underline the potential for eukaryotic cell-free systems to significantly improve drug development pipelines.

Cell-free protein synthesis as a novel tool for directed glycoengineering of active erythropoietin

As one of the most complex post-translational modification, glycosylation is widely involved in cell adhesion, cell proliferation and immune response. Nevertheless glycoproteins with an identical polypeptide backbone mostly differ in their glycosylation patterns. Due to this heterogeneity, the mapping of different glycosylation patterns to their associated function is nearly impossible. In the last years, glycoengineering tools including cell line engineering, chemoenzymatic remodeling and site-specific glycosylation have attracted increasing interest. The therapeutic hormone erythropoietin (EPO) has been investigated in particular by various groups to establish a production process resulting in a defined glycosylation pattern. However commercially available recombinant human EPO shows batch-to-batch variations in its glycoforms. Therefore we present an alternative method for the synthesis of active glycosylated EPO with an engineered O-glycosylation site by combining eukaryotic cell-free protein synthesis and site-directed incorporation of non-canonical amino acids with subsequent chemoselective modifications.

Energizing eukaryotic cell-free protein synthesis with glucose metabolism

Eukaryotic cell-free protein synthesis (CFPS) is limited by the dependence on costly high-energy phosphate compounds and exogenous enzymes to power protein synthesis (e.g., creatine phosphate and creatine kinase, CrP/CrK). Here, we report the ability to use glucose as a secondary energy substrate to regenerate ATP in a Saccharomyces cerevisiae crude extract CFPS platform. We observed synthesis of 3.64±0.35μgmL−1 active luciferase in batch reactions with 16mM glucose and 25mM phosphate, resulting in a 16% increase in relative protein yield (μg protein/$ reagents) compared to the CrP/CrK system. Our demonstration provides the foundation for development of cost-effective eukaryotic CFPS platforms.

Characterizing IGR IRES-mediated translation initiation for use in yeast cell-free protein synthesis

Eukaryotic cell-free protein synthesis (CFPS) systems are limited, in part, by inefficient translation initiation. Here, we report three internal ribosome entry site (IRES) sequences from the Dicistroviridae family that are highly active in yeast CFPS. These include the intergenic region (IGR) IRES from cricket paralysis virus (CrPV), plautia stali intestine virus (PSIV) and Solenopsis invicta virus 1 (SINV1). Optimization of combined transcription and translation (Tx/Tl) CFPS reactions primed with linear DNA containing the CrPV IGR IRES resulted in batch synthesis yields of 0.92 ± 0.17 μg/mL luciferase. Further template engineering, such as including the first 12 nt of native CrPV gene, increased yields to 2.33 ± 0.11 μg/mL. We next observed that the inclusion of a 50 nt poly(A) to the 3' end of the IGR IRES-mediated message increased yields an additional 81% to 4.33 ± 0.37 μg/mL, without any effect on mRNA stability or copy number. This was surprising because the CrPV IGR IRES requires no known translation initiation factors. Lastly, we investigated a method to inhibit background expression through competitive inhibition by supplying the reaction with 5' cap structure analog. This study highlights the crucial role translation initiation plays in yeast CFPS and offers a simple platform to study IRES sequences.

Efficiency of cell-free protein synthesis based on a crude cell extract from Escherichia coli, wheat germ, and rabbit reticulocytes

The efficiency of protein synthesis for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was examined with several in vitro coupled transcription/translation protein synthesis systems based on Escherichia coli lysate, wheat germ, or reticulocyte lysate, and an in vitro translation system based on wheat germ extract. A significant amount of protein synthesis was observed only in systems based on E. coli using pET/G3PDH as the expression vector. A remarkable increase of protein synthesis was obtained in wheat germ using a pT NT expression vector which contains a 5′-globin leader sequence and a synthetic poly(A) 30 tail instead of pET. A significant difference of T7 RNA polymerase presence by Western blot analysis was not observed in the first four systems, and the difference of total RNA presence in each reaction mixture by Northern blot analysis seemed unrelated to protein synthesis. Although a small amount of protein was synthesized using RNA-encoding G3PDH transcribed in vitro with pET/G3PDH by an in vitro translation system, an extreme increase was observed using transcribed RNA with pEU/G3PDH, which contains T7 RNA promoter and a translation enhancer, Ω sequence. These results suggest that the presence of an enhancer sequence for translation is one of the critical steps for protein synthesis by a eukaryotic cell-free protein synthesis system.

Fluorescent labeling of cell-free synthesized proteins by incorporation of fluorophore-conjugated nonnatural amino acids

Although fluorescent dyes, such as fluorescein derivatives, have bulky and complex structures, nonnatural amino acids carrying these fluorescein derivatives are acceptable by the Escherichia coli ribosome and are useful for the cotranslational fluorescent labeling of cell-free synthesized proteins. Surprisingly, the incorporation efficiency of nonnatural amino acids carrying fluorescein derivatives into translated proteins depends on the source of the translational machinery used in cell-free protein synthesis. That is, whereas the E. coli ribosome efficiently supported the incorporation of nonnatural amino acids carrying fluorescein derivatives into a protein structure, no detectable fluorescent signal was observed from the protein expressed in the eukaryotic cell-free protein synthesis system performed in the presence of fluorescein-conjugated aminoacylated transfer RNA (tRNA).

Inhibition of eukaryotic cell-free protein synthesis by thionins from wheat endosperm

Thionins are polypeptide toxins of about 5000 molecular weight, present in the endosperms of many Gramineae, which modify membrane permeability and inhibit macromolecular synthesis in cultured mammalian cells. Evidence is presented that they inhibit in vitro protein synthesis at micromolar concentrations in cell-free systems derived from wheat germ or from rabbit reticulocytes. Inhibition seems to occur by direct binding of mRNA by the toxin, as judged by the ability of thionins to mediate retention of RNA in nitrocellulose filters and by the dependence of inhibitory concentrations on the amount of exogenous RNA added to the wheat-germ translation system. Commercial preparations of wheat-germ have been found to include some endosperm contamination (up to 15%), which may result in at least partially inhibitory concentrations of the toxin in the cell-free extracts.