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 - 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 -