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Rapid prototyping. Green Fluorescent Proteins. Cell Survival. Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology. Journal of visualized experiments : JoVE , Levine, Max Z. In: Journal of visualized experiments : JoVE. AU - Gregorio, Nicole E. AU - Oza, Javin P. Journal of visualized experiments : JoVE. The CHO cell-free system contains endogenous microsomes derived from the endoplasmic reticulum, which enables a direct integration of membrane proteins into a nature like milieu and the introduction of posttranslational modifications.

Different steps of system development are described including the cultivation of CHO cells, cell harvesting and cell disruption to prepare translationally active CHO cell lysates. The requirements for DNA templates and the generation of linear DNA templates suitable for the CHO cell-free reaction is further depicted to underline the opportunity to produce different protein variants in a short period. This experimental setup provides a basis for high-throughput applications.

In this way, a direct interaction of the IRES structure with the ribosome facilitates a translation factor independent initiation of translation.

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The reaction format was further adjusted to a continuous exchange CHO cell-free reaction CHO CECF to prolong reaction time and thereby increase the productivity of the cell-free systems. The CHO CECF system represents a sophisticated resource to address structural and functional aspects of difficult-to-express proteins in fundamental and applied research.

In vitro protein production, Internal ribosomal entry sites, Continuous exchange cell-free systems, Difficult-to-express proteins, Linear expression templates. Antibody Data Search Beta. Authors: Lena Thoring 1 ,. Stefan Kubick 1. Lena Thoring 1 ,. Full text PDF Related articles. Abstract We present an alternative production platform for the synthesis of complex proteins. Transfusion 52 , — Parsa, C.

A novel protective effect of erythropoietin in the infarcted heart. Journal of Clinical Investigation , — Gaddam, S. Kittur, F.

The Production Of Human Leptin Using Cell Free Protein Synthesis

Skibeli, V. Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin. Blood 98 , Sachse, R. Synthesis of membrane proteins in eukaryotic cell-free systems. Life Sci. Quast, R. Synthesis and site-directed fluorescence labeling of azido proteins using eukaryotic cell-free orthogonal translation systems.

Analytical Biochemistry , 4—9 Kubick, S. Stech, M. Cell-free eukaryotic systems for the production, engineering, and modification of scFv antibody fragments.

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Kolarich, D. Determination of site-specific glycan heterogeneity on glycoproteins. Protocols 7 , — Schachter, H. Paucimannose N-glycans in Caenorhabditis elegans and Drosophila melanogaster. Kamerling , — Komatsu, N. Cancer Res 51 , Davis, T. Comparison of oligosaccharide processing among various insect cell lines expressing a secreted glycoprotein. Kawar, Z. Glycobiology 7 , — Stanley, P. Essentials of Glycobiology. Altmann, F. Processing of asparagine-linked oligosaccharides in insect cells.

N-Acetylglucosaminyltransferase I and II activities in cultured lepidopteran cells. Glycobiology 3 , — Tenno, M. Molecular and Cellular Biology 27 , — Opat, A. Trafficking and localisation of resident Golgi glycosylation enzymes. Biochimie 83 , — Gong, B.

Glycosylation characterization of recombinant human erythropoietin produced in glycoengineered Pichia pastoris by mass spectrometry. Mass Spectrom. Lalonde, M. Therapeutic glycoprotein production in mammalian cells. Journal of Biotechnology , — Khan, A. Humanizing glycosylation pathways in eukaryotic expression systems. World Journal of Microbiology and Biotechnology 33 , 4 Yang, Q.

ACS Chem. Kim, Y. Production and N-glycan analysis of secreted human erythropoietin glycoprotein in stably transfected Drosophila S2 cells. Wang, Y. Efficient preparation and PEGylation of recombinant human non-glycosylated erythropoietin expressed as inclusion body in E. International Journal of Pharmaceutics , — Jeong, T.

Soluble expression and partial purification of recombinant human erythropoietin from E. Protein Expression and Purification 95 , — Choudhury, A. Evaluating fermentation effects on cell growth and crude extract metabolic activity for improved yeast cell-free protein synthesis. Biochemical Engineering Journal 91 , — Scientific Reports 6 , EP Zawada, J.

Biotechnology and Bioengineering , — Albayrak, C. Using E. Biochemical and Biophysical Research Communications , — Egrie, J. Glycoconjugate Journal 10 , Development and characterization of novel erythropoiesis stimulating protein NESP. Br J Cancer 84 , 3—10 Macdougall, I. Clinical Journal of the American Society of Nephrology 3 , — Matthews, D. A sequential dimerization mechanism for erythropoietin receptor activation. Syed, R. Efficiency of signalling through cytokine receptors depends critically on receptor orientation.

Nature , EP Wen, D. Erythropoietin structure-function relationships. Identification of functionally important domains. Blood 89 , Pankratova, S. Neuroprotective properties of a novel, non-haematopoietic agonist of the erythropoietin receptor. Brain , — Martin, R.

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ACS Synth. Boissel, J. Mutant proteins that test a model of tertiary structure. Depaolis, A.

Protocols for a robust, flexible, and accessible platform technology

Characterization of erythropoietin dimerization. Sytkowski, A. Human erythropoietin dimers with markedly enhanced in vivo activity. Hoffmann, E. Stabilization of bacterially expressed erythropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylation sites. Download references. We thank Prof. Wolfgang Jabs for the careful revision of the mass spectrometry data. Correspondence to Stefan Kubick.

An Introduction to Cell-Free Expression

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If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Advanced search. Skip to main content. Subjects Chemical modification Glycobiology. Abstract As one of the most complex post-translational modification, glycosylation is widely involved in cell adhesion, cell proliferation and immune response.

Materials and Methods Lysate preparation Translationally active insect cell lysates were produced as described previously Protein quantification using hot TCA precipitation The yield of cell-free synthesized EPO was determined by hot trichloroacetic acid TCA precipitation and liquid scintillation quantification as described previously Figure 1. Full size image. Figure 2.