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 - 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 - 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 -