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 - 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 - Haueis, Lisa A1 - Stech, Marlitt A1 - Kubick, Stefan T1 - A Cell-free Expression Pipeline for the Generation and Functional Characterization of Nanobodies JF - Frontiers in Bioengineering and Biotechnology N2 - Cell-free systems are well-established platforms for the rapid synthesis, screening, engineering and modification of all kinds of recombinant proteins ranging from membrane proteins to soluble proteins, enzymes and even toxins. Also within the antibody field the cell-free technology has gained considerable attention with respect to the clinical research pipeline including antibody discovery and production. Besides the classical full-length monoclonal antibodies (mAbs), so-called "nanobodies" (Nbs) have come into focus. A Nb is the smallest naturally-derived functional antibody fragment known and represents the variable domain (VHH, similar to 15 kDa) of a camelid heavy-chain-only antibody (HCAb). Based on their nanoscale and their special structure, Nbs display striking advantages concerning their production, but also their characteristics as binders, such as high stability, diversity, improved tissue penetration and reaching of cavity-like epitopes. The classical way to produce Nbs depends on the use of living cells as production host. Though cell-based production is well-established, it is still time-consuming, laborious and hardly amenable for high-throughput applications. Here, we present for the first time to our knowledge the synthesis of functional Nbs in a standardized mammalian cell-free system based on Chinese hamster ovary (CHO) cell lysates. Cell-free reactions were shown to be time-efficient and easy-to-handle allowing for the "on demand" synthesis of Nbs. Taken together, we complement available methods and demonstrate a promising new system for Nb selection and validation. KW - cell-free protein synthesis KW - In vitro transcription KW - translation KW - nanobody KW - VHH KW - camelid KW - CHO cell lysate Y1 - 2022 U6 - https://doi.org/10.3389/fbioe.2022.896763 SN - 2296-4185 VL - 10 PB - Frontiers Media CY - Lausanne 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 - Wüstenhagen, Doreen Anja A1 - Lukas, Phil A1 - Müller, Christian A1 - Aubele, Simone A. A1 - Hildebrandt, Jan-Peter A1 - Kubick, Stefan T1 - Cell-free synthesis of the hirudin variant 1 of the blood-sucking leech Hirudo medicinalis JF - Scientific reports N2 - Synthesis and purification of peptide drugs for medical applications is a challenging task. The leech-derived factor hirudin is in clinical use as an alternative to heparin in anticoagulatory therapies. So far, recombinant hirudin is mainly produced in bacterial or yeast expression systems. We describe the successful development and application of an alternative protocol for the synthesis of active hirudin based on a cell-free protein synthesis approach. Three different cell lysates were compared, and the effects of two different signal peptide sequences on the synthesis of mature hirudin were determined. The combination of K562 cell lysates and the endogenous wild-type signal peptide sequence was most effective. Cell-free synthesized hirudin showed a considerably higher anti-thrombin activity compared to recombinant hirudin produced in bacterial cells. Y1 - 2020 U6 - https://doi.org/10.1038/s41598-020-76715-w SN - 2045-2322 VL - 10 IS - 1 PB - Macmillan Publishers Limited, part of Springer Nature CY - London ER - 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 - 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 - GEN A1 - Zemella, Anne A1 - Thoring, Lena A1 - Hoffmeister, Christian A1 - Šamalíková, Mária 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 T2 - Postprints der Universität Potsdam : Mathematisch Naturwissenschaftliche Reihe 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 824 KW - recombinat-human-erythropoietin KW - glycosylation KW - expression KW - site KW - anemia KW - CDNA KW - glycoprotein KW - purification KW - cloning KW - growth Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-427017 IS - 824 ER - TY - JOUR A1 - Scheller, Frieder W. A1 - Yarman, Aysu A1 - Bachmann, Till A1 - Hirsch, Thomas A1 - Kubick, Stefan A1 - Renneberg, Reinhard A1 - Schumacher, Soeren A1 - Wollenberger, Ursula A1 - Teller, Carsten A1 - Bier, Frank Fabian ED - Gu, MB ED - Kim, HS T1 - Future of biosensors: a personal view JF - Advances in biochemical engineering, biotechnology JF - Advances in Biochemical Engineering-Biotechnology N2 - Biosensors representing the technological counterpart of living senses have found routine application in amperometric enzyme electrodes for decentralized blood glucose measurement, interaction analysis by surface plasmon resonance in drug development, and to some extent DNA chips for expression analysis and enzyme polymorphisms. These technologies have already reached a highly advanced level and need minor improvement at most. The dream of the "100-dollar' personal genome may come true in the next few years provided that the technological hurdles of nanopore technology or of polymerase-based single molecule sequencing can be overcome. Tailor-made recognition elements for biosensors including membrane-bound enzymes and receptors will be prepared by cell-free protein synthesis. As alternatives for biological recognition elements, molecularly imprinted polymers (MIPs) have been created. They have the potential to substitute antibodies in biosensors and biochips for the measurement of low-molecular-weight substances, proteins, viruses, and living cells. They are more stable than proteins and can be produced in large amounts by chemical synthesis. Integration of nanomaterials, especially of graphene, could lead to new miniaturized biosensors with high sensitivity and ultrafast response. In the future individual therapy will include genetic profiling of isoenzymes and polymorphic forms of drug-metabolizing enzymes especially of the cytochrome P450 family. For defining the pharmacokinetics including the clearance of a given genotype enzyme electrodes will be a useful tool. For decentralized online patient control or the integration into everyday "consumables' such as drinking water, foods, hygienic articles, clothing, or for control of air conditioners in buildings and cars and swimming pools, a new generation of "autonomous' biosensors will emerge. KW - Biosensors KW - Molecularly imprinted polymers KW - Personalized medicine Y1 - 2014 SN - 978-3-642-54143-8; 978-3-642-54142-1 U6 - https://doi.org/10.1007/10_2013_251 SN - 0724-6145 VL - 140 SP - 1 EP - 28 PB - Springer CY - Berlin ER - TY - JOUR A1 - Sachse, Rita A1 - Wüstenhagen, Doreen Anja A1 - Samalikova, Maria A1 - Gerrits, Michael A1 - Bier, Frank Fabian A1 - Kubick, Stefan T1 - Synthesis of membrane proteins in eukaryotic cell-free systems JF - Engineering in life sciences : Industry, Environment, Plant, Food N2 - Cell-free protein synthesis (CFPS) is a valuable method for the fast expression of difficult-to-express proteins as well as posttranslationally modified proteins. Since cell-free systems circumvent possible cytotoxic effects caused by protein overexpression in living cells, they significantly enlarge the scale and variety of proteins that can be characterized. We demonstrate the high potential of eukaryotic CFPS to express various types of membrane proteins covering a broad range of structurally and functionally diverse proteins. Our eukaryotic cell-free translation systems are capable to provide high molecular weight membrane proteins, fluorescent-labeled membrane proteins, as well as posttranslationally modified proteins for further downstream analysis. KW - Cell-free protein expression KW - In vitro protein synthesis KW - Labeled membrane proteins KW - Synthetic glycoprotein Y1 - 2013 U6 - https://doi.org/10.1002/elsc.201100235 SN - 1618-0240 VL - 13 IS - 1 SP - 39 EP - 48 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Stech, Marlitt A1 - Merk, Helmut A1 - Schenk, Jörg A. A1 - Stöcklein, Walter F. M. A1 - Wüstenhagen, Doreen Anja A1 - Micheel, Burkhard A1 - Duschl, Claus A1 - Bier, Frank Fabian A1 - Kubick, Stefan T1 - Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system JF - Journal of biotechnology N2 - Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates. These lysates contain translocationally active microsomal structures derived from the endoplasmic reticulum (ER), allowing for posttranslational modifications of cell-free synthesized proteins. Both types of these insect cell lysates enable the synthesis and translocation of scFv into ER-derived vesicles. However, only the one that has a specifically adapted redox potential yields functional active antibody fragments. We have developed a new methodology for the isolation of functional target proteins based on the translocation of cell-free produced scFv into microsomal structures and subsequent collection of protein-enriched vesicles. Antibody fragments that have been released from these vesicles are shown to be well suited for label-free binding studies. Altogether, these results show the potential of insect cell lysates for the production, purification and selection of antibody fragments in an easy-to-handle and time-saving manner. KW - Cell-free KW - In vitro translation KW - Single chain antibody (scFv) KW - Insect lysate KW - Surface plasmon resonance Y1 - 2012 U6 - https://doi.org/10.1016/j.jbiotec.2012.08.020 SN - 0168-1656 VL - 164 IS - 2 SP - 220 EP - 231 PB - Elsevier CY - Amsterdam ER -