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.
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Versatile Cell-Free Protein Synthesis Systems Based on Chinese Hamster Ovary Cells
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Cell-Free Protein Expression | Thermo Fisher Scientific - US
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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.