title: Cell-free protein synthesis systems derived from cultured mammalian cells.
abstract: We present a technology for the production of target proteins using novel cell-free systems derived from cultured human K562 cells and Chinese hamster ovary (CHO) cells. The protocol includes the cultivation of cells, the preparation of translationally active lysates, and the cell-free synthesis of desired proteins. An efficient expression vector based on the internal ribosome entry site (IRES) from the intergenic region (IGR) of the cricket paralysis virus (CrPV) was constructed for both systems. The coupled batch-based platforms enable the synthesis of a broad range of target proteins such as cytosolic proteins, secreted proteins, membrane proteins embedded into endogenous microsomes, and glycoproteins. The glycosylation of erythropoietin demonstrates the successful performance of posttranslational modifications in the novel cell-free systems. Protein yields of approximately 20 μg/ml (K562-based cell-free system) and 50 μg/ml (CHO-based cell-free system) of active firefly luciferase are obtained in the coupled transcription-translation systems within 3 h. As a result, both cell-free protein synthesis systems serve as powerful tools for high-throughput proteomics.
authors.affiliation: Department of Cell-free Bioproduction, Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses Potsdam-Golm (IZI-BB), Am Mühlenberg 13, 14476, Potsdam, Germany.
authors.forename: Andreas K
authors.initials: AK
authors.lastname: Brödel
authors.forename: Doreen A
authors.initials: DA
authors.lastname: Wüstenhagen
authors.forename: Stefan
authors.initials: S
authors.lastname: Kubick
mesh_terms: D000818:Animals; D001709:Biotechnology / Q000379:methods*; D016466:CHO Cells; D018929:Cell Culture Techniques; D002474:Cell-Free System / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D014780:Viruses / Q000235:genetics*
chemical_list: D011506:Proteins