TY - JOUR A1 - Dondapati, Srujan Kumar A1 - Lübberding, Henning A1 - Zemella, Anne A1 - Thoring, Lena A1 - Wüstenhagen, Doreen Anja A1 - Kubick, Stefan T1 - Functional Reconstitution of Membrane Proteins Derived From Eukaryotic Cell-Free Systems JF - Frontiers in pharmacology N2 - Cell-free protein synthesis (CFPS) based on eukaryotic Sf21 lysate is gaining interest among researchers due to its ability to handle the synthesis of complex human membrane proteins (MPs). Additionally Sf21 cell-free systems contain endogenous microsomal vesicles originally derived from the endoplasmic reticulum (ER). After CFPS, MPs will be translocated into the microsomal vesicles membranes present in the lysates. Thus microsomal membranes offer a natural environment for de novo synthesized MPs. Despite the advantage of synthesizing complex MPs with post translational modifications directly into the microsomal membranes without any additional solubilization supplements, batch based Sf21 cell-free synthesis suffers from low yields. The bottleneck for MPs in particular after the synthesis and incorporation into the microsomal membranes is to analyze their functionality. Apart from low yields of the synthesized MPs with batch based cell-free synthesis, the challenges arise in the form of cytoskeleton elements and peripheral endogenous proteins surrounding the microsomes which may impede the functional analysis of the synthesized proteins. So careful sample processing after the synthesis is particularly important for developing the appropriate functional assays. Here we demonstrate how MPs (native and batch synthesized) from ER derived microsomes can be processed for functional analysis by electrophysiology and radioactive uptake assay methods. Treatment of the microsomal membranes either with a sucrose washing step in the case of human serotonin transporter (hSERT) and sarco/endoplasmic reticulum Ca2+/ATPase (SERCA) pump or with mild detergents followed by the preparation of proteoliposomes in the case of the human voltage dependent anionic channel (hVDAC1) helps to analyze the functional properties of MPs. KW - membrane proteins KW - Sf21 lysates KW - microsomes KW - cell-free protein synthesis KW - proteoliposomes KW - transporter KW - ion channel KW - pump Y1 - 2019 U6 - https://doi.org/10.3389/fphar.2019.00917 SN - 1663-9812 VL - 10 PB - Frontiers Research Foundation CY - Lausanne ER - TY - JOUR A1 - Ramm, Franziska A1 - Jack, Lena A1 - Kaser, Danny A1 - Schloßhauer, Jeffrey L. A1 - Zemella, Anne A1 - Kubick, Stefan T1 - Cell-Free Systems Enable the Production of AB(5) Toxins for Diagnostic Applications JF - Toxins N2 - 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 AB(5) 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 AB(5) 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 AB(5) 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. KW - cholera toxin KW - heat-labile enterotoxin KW - AB(5) toxins KW - eukaryotic KW - cell-free systems KW - orthogonal systems Y1 - 2022 U6 - https://doi.org/10.3390/toxins14040233 SN - 2072-6651 VL - 14 IS - 4 PB - MDPI CY - Basel ER - TY - JOUR A1 - Zemella, Anne A1 - Thoring, Lena A1 - Hoffmeister, Christian A1 - Samalikova, Maria A1 - Ehren, Patricia A1 - Wüstenhagen, Doreen Anja A1 - Kubick, Stefan T1 - Cell-free protein synthesis as a novel tool for directed glycoengineering of active erythropoietin JF - Scientific reports N2 - 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. Y1 - 2018 U6 - https://doi.org/10.1038/s41598-018-26936-x SN - 2045-2322 VL - 8 PB - Nature Publ. Group CY - London ER - TY - JOUR A1 - Dondapati, Srujan Kumar A1 - Stech, Marlitt A1 - Zemella, Anne A1 - Kubick, Stefan T1 - Cell-free protein synthesis BT - a promising option for future drug development JF - BioDrugs N2 - 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. Y1 - 2020 U6 - https://doi.org/10.1007/s40259-020-00417-y SN - 1173-8804 SN - 1179-190X VL - 34 IS - 3 SP - 327 EP - 348 PB - Springer CY - Northcote ER - TY - JOUR A1 - Schloßhauer, Jeffrey A1 - Cavak, Niño A1 - Zemella, Anne A1 - Thoring, Lena A1 - Kubick, Stefan T1 - Cell engineering and cultivation of chinese hamster ovary cells for the development of orthogonal eukaryotic cell-free translation systems JF - Frontiers in molecular biosciences N2 - 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. KW - orthogonal translation KW - cell-free protein synthesis KW - CRISPR KW - amber suppression KW - E. coli tyrosyl-tRNA synthetase KW - M. mazei pyrrolysyl-tRNA synthetase KW - membrane protein KW - C12orf35 Y1 - 2022 U6 - https://doi.org/10.3389/fmolb.2022.832379 SN - 2296-889X VL - 9 PB - Frontiers Media CY - Lausanne ER - TY - JOUR A1 - Krebs, Simon K. A1 - Rakotoarinoro, Nathanael A1 - Stech, Marlitt A1 - Zemella, Anne A1 - Kubick, Stefan T1 - A CHO-based cell-free dual fluorescence reporter system for the straightforward assessment of amber suppression and scFv functionality JF - Frontiers in Bioengineering and Biotechnology N2 - Incorporation of noncanonical amino acids (ncAAs) with bioorthogonal reactive groups by amber suppression allows the generation of synthetic proteins with desired novel properties. Such modified molecules are in high demand for basic research and therapeutic applications such as cancer treatment and in vivo imaging. The positioning of the ncAA-responsive codon within the protein's coding sequence is critical in order to maintain protein function, achieve high yields of ncAA-containing protein, and allow effective conjugation. Cell-free ncAA incorporation is of particular interest due to the open nature of cell-free systems and their concurrent ease of manipulation. In this study, we report a straightforward workflow to inquire ncAA positions in regard to incorporation efficiency and protein functionality in a Chinese hamster ovary (CHO) cell-free system. As a model, the well-established orthogonal translation components Escherichia coli tyrosyl-tRNA synthetase (TyrRS) and tRNATyr(CUA) were used to site-specifically incorporate the ncAA p-azido-l-phenylalanine (AzF) in response to UAG codons. A total of seven ncAA sites within an anti-epidermal growth factor receptor (EGFR) single-chain variable fragment (scFv) N-terminally fused to the red fluorescent protein mRFP1 and C-terminally fused to the green fluorescent protein sfGFP were investigated for ncAA incorporation efficiency and impact on antigen binding. The characterized cell-free dual fluorescence reporter system allows screening for ncAA incorporation sites with high incorporation efficiency that maintain protein activity. It is parallelizable, scalable, and easy to operate. We propose that the established CHO-based cell-free dual fluorescence reporter system can be of particular interest for the development of antibody-drug conjugates (ADCs). KW - expanded genetic code KW - orthogonal system KW - noncanonical amino acid KW - unnatural amino acid KW - antibody KW - cell-free protein synthesis KW - mRFP1 KW - sfGFP Y1 - 2022 U6 - https://doi.org/10.3389/fbioe.2022.873906 SN - 2296-4185 VL - 10 PB - Frontiers Media CY - Lausanne ER -