Profiling Cellular Morphological Changes Induced by Dual-Targeting PROTACs of Aurora Kinase and RNA-Binding Protein YTHDF2.

Proteolysis targeting chimeras (PROTACs) are new chemical modalities that degrade proteins of interest, including established kinase targets and emerging RNA-binding proteins (RBPs). Whereas diverse sets of biochemical, biophysical and cellular assays are available for the evaluation and optimizations of PROTACs in understanding the involved ubiquitin-proteasome-mediated degradation mechanism and the structure-degradation relationship, a phenotypic method profiling the cellular morphological changes is rarely used. In this study, first, we reported the only examples of PROTACs degrading the mRNA-binding protein YTHDF2 via screening of multikinase PROTACs. Second, we reported the profiling of cellular morphological changes of the dual kinase- and RBP-targeting PROTACs using the unbiased cell painting assay (CPA). The CPA analysis revealed the high biosimilarity with the established aurora kinase cluster and annotated aurora kinase inhibitors, which reflected the association between YTHDF2 and the aurora kinase signaling network. Broadly, the results demonstrated that the cell painting assay can be a straightforward and powerful approach to evaluate PROTACs. Complementary to the existing biochemical, biophysical and cellular assays, CPA provided a new perspective in characterizing PROTACs at the cellular morphology.

A Cost-Effective Pichia pastoris Cell-Free System Driven by Glycolytic Intermediates Enables the Production of Complex Eukaryotic Proteins.

Cell-free systems are particularly attractive for screening applications and the production of difficult-to-express proteins. However, the production of cell lysates is difficult to implement on a larger scale due to large time requirements, cultivation costs, and the supplementation of cell-free reactions with energy regeneration systems. Consequently, the methylotrophic yeast Pichia pastoris, which is widely used in recombinant protein production, was utilized in the present study to realize cell-free synthesis in a cost-effective manner. Sensitive disruption conditions were evaluated, and appropriate signal sequences for translocation into ER vesicles were identified. An alternative energy regeneration system based on fructose-1,6-bisphosphate was developed and a ~2-fold increase in protein production was observed. Using a statistical experiment design, the optimal composition of the cell-free reaction milieu was determined. Moreover, functional ion channels could be produced, and a G-protein-coupled receptor was site-specifically modified using the novel cell-free system. Finally, the established P. pastoris cell-free protein production system can economically produce complex proteins for biotechnological applications in a short time.

Promoting the production of challenging proteins via induced expression in CHO cells and modified cell-free lysates harboring T7 RNA polymerase and mutant eIF2α.

Chinese hamster ovary (CHO) cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications. However, toxic proteins and membrane proteins are often difficult-to-express in living cells. Alternatively, cell-free protein synthesis can be employed. This study explores innovative strategies for enhancing the production of challenging proteins through the modification of CHO cells by investigating both, cell-based and cell-free approaches. A major result in our study involves the integration of a mutant eIF2 translation initiation factor and T7 RNA polymerase into CHO cell lysates for cell-free protein synthesis. This resulted in elevated yields, while eliminating the necessity for exogenous additions during cell-free production, thereby substantially enhancing efficiency. Additionally, we explore the potential of the Rosa26 genomic site for the integration of T7 RNA polymerase and cell-based tetracycline-controlled protein expression. These findings provide promising advancements in bioproduction technologies, offering flexibility to switch between cell-free and cell-based protein production as needed.

Just Keep Rolling?-An Encompassing Review towards Accelerated Vaccine Product Life Cycles.

In light of the recent pandemic, several COVID-19 vaccines were developed, tested and approved in a very short time, a process that otherwise takes many years. Above all, these efforts have also unmistakably revealed the capacity limits and potential for improvement in vaccine production. This review aims to emphasize recent approaches for the targeted rapid adaptation and production of vaccines from an interdisciplinary, multifaceted perspective. Using research from the literature, stakeholder analysis and a value proposition canvas, we reviewed technological innovations on the pharmacological level, formulation, validation and resilient vaccine production to supply bottlenecks and logistic networks. We identified four main drivers to accelerate the vaccine product life cycle: computerized candidate screening, modular production, digitized quality management and a resilient business model with corresponding transparent supply chains. In summary, the results presented here can serve as a guide and implementation tool for flexible, scalable vaccine production to swiftly respond to pandemic situations in the future.

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.

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.

Cell Engineering and Cultivation of Chinese Hamster Ovary Cells for the Development of Orthogonal Eukaryotic Cell-free Translation Systems.

The investigation of protein structures, functions and interactions often requires modifications to adapt protein properties to the specific application. Among many possible methods to equip proteins with new chemical groups, the utilization of orthogonal aminoacyl-tRNA synthetase/tRNA pairs enables the site-specific incorporation of non-canonical amino acids at defined positions in the protein. The open nature of cell-free protein synthesis reactions provides an optimal environment, as the orthogonal components do not need to be transported across the cell membrane and the impact on cell viability is negligible. In the present work, it was shown that the expression of orthogonal aminoacyl-tRNA synthetases in CHO cells prior to cell disruption enhanced the modification of the pharmaceutically relevant adenosine A2a receptor. For this purpose, in complement to transient transfection of CHO cells, an approach based on CRISPR/Cas9 technology was selected to generate a translationally active cell lysate harboring endogenous orthogonal aminoacyl-tRNA synthetase.

Cell-Free Systems Enable the Production of AB5 Toxins for Diagnostic Applications.

Cell-free protein synthesis (CFPS) represents a versatile key technology for the production of toxic proteins. As a cell lysate, rather than viable cells, is used, the toxic effects on the host organism can be circumvented. The open nature of cell-free systems allows for the addition of supplements affecting protein concentration and folding. Here, we present the cell-free synthesis and functional characterization of two AB5 toxins, namely the cholera toxin (Ctx) and the heat-labile enterotoxin (LT), using two eukaryotic cell-free systems based on Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cells. Through an iterative optimization procedure, the synthesis of the individual AB5 toxins was established, and the formation of multimeric structures could be shown by autoradiography. A functional analysis was performed using cell-based assays, thereby demonstrating that the LT complex induced the characteristic cell elongation of target cells after 24 h. The LT complex induced cell death at higher concentrations, starting at an initial concentration of 5 nM. The initial toxic effects of the Ctx multimer could already be detected at 4 nM. The detection and characterization of such AB5 toxins is of utmost importance, and the monitoring of intracellular trafficking facilitates the further identification of the mechanism of action of these toxins. We showed that the B-subunit of LT (LTB) could be fluorescently labeled using an LTB-Strep fusion protein, which is a proof-of-concept for future Trojan horse applications. Further, we performed a mutational analysis of the CtxA subunit as its template was modified, and an amber stop codon was inserted into CtxA's active site. Subsequently, a non-canonical amino acid was site-specifically incorporated using bio-orthogonal systems. Finally, a fluorescently labeled CtxA protein was produced using copper-catalyzed click reactions as well as a Staudinger ligation. As expected, the modified Ctx multimer no longer induced toxic effects. In our study, we showed that CFPS could be used to study the active centers of toxins by inserting mutations. Additionally, this methodology can be applied for the design of Trojan horses and targeted toxins, as well as enabling the intracellular trafficking of toxins as a prerequisite for the analysis of the toxin's mechanism of action.