Eukaryotic expression

Conclusion. We have expressed eukaryotic CK in . coli and have demonstrated that active protein can be recovered from the resulting inclusion bodies. Based on the parameters tested, this protein appears to be very similar to CK purified from electric organ tissue. The recovery scheme is simple, involving cell lysis and overnight extraction with a detergent-containing buffer followed by sonication and isolation of... 

Breathnach R., Chambon P. (1981) Organization and expression eukaryotic of split genes for coding proteins. Annu. 

Jiang, Y., Vasconcelles, M.J., Wretzel, S., Light, A., Gilooly, L., McDaid, K., Oh, C.S., Martin, C.E. and Goldberg, M.A. Mga2p processing by hypoxia and unsaturated fatty acids in Saccharomyces cerevisiae impact on LORE-dependent gene expression. Eukaryot Cell, 1 (2002) 481-490. 

For higher eukaryotes, the most common approach to cloning is PCR amplification from cDNA libraries in order to avoid the intron problem. Another challenge when expressing eukaryotic proteins is that eukaryotic proteins frequently do not fold properly in E. coli and will not be processed with the correct post-translational modifications, and therefore often other eukaryotic expression hosts must be used. Options include using yeast cells, insect cell lines or mammalian cell lines. 

Currently, CHO cells are the most widely used technology in biomanufacturing because they are capable of expressing eukaryotic proteins (processing, folding, and post-translational modifications) that cannot be provided by E. coli. A long... 

Cleary, M. D., Singh, U., Blader, 1. J., Brewer, J. L., and Boothroyd, J. C., 2002, Toxoplasma gondii asexual development identification of developmentaUy regulated genes and distinct patterns of gene expression. Eukaryot Cell 1 329-340. 

Lodha, M., Schulz-Raffelt, M., and Schroda, M. (2008) A new assay for promoter analysis in Chlamydomonas reveals roles for heat shock elements and the TATA box in HSP70A promoter-mediated activation of transgene expression. Eukaryotic Cell, 7 (1), 172-176. 

Because different cell types in eukaryotic organisms express selected subsets of genes, RNA preparations from cells or tissues in which genes of interest are selectively transcribed are enriched for the desired mRNAs. cDNA... 

The Ca2+-binding subunit TN-C is homologous to calmodulin with four EF-hands. In contrast to calmodulin, which is ubiquitously expressed in multicellular eukaryotic organisms and interacts with many targets, troponin specifically regulates muscle contraction. There are some structural differences between Troponin C in skeletal and cardiac muscles reflecting their physiological differences. 

The field of DNA vaccination started when eukaryotic expression vectors were injected into the muscle of laboratory animals [2]. The authors observed protein expression for more than 2 months after injection and noted that no special delivery system was required to obtain this expression. Subsequently, it was demonstrated that antibodies can be induced simply by injecting plasmid DNA into the muscle of mice [3]. Subsequent studies found that the injection of expression plasmids also leads to the induction of a cytotoxic T-cell response. After injection, the DNA enters cells of the vaccinated host and the encoded gene becomes expressed. This eventually leads to the induction of a cellular cytotoxic T-cell, T-helper, and/or humoral (antibody) immune response. 

There are various protocols of administering eukaryotic expression vectors aiming to deliver (i.e. transfect) the DNA into the cytoplasm of the host cells (see Figure 2). The DNA is subsequently imported into the nucleus of the transfected cells allowing expression of the... 

The eukaryotic expression cassette is the part of an expression vector that enables production of a protein in a eukaryotic cell. The cassette consists of a eukaryotic promoter for mRNA transcription, the gene and an mRNA termination and processing signal (Poly-A signal). 

Heterologous expression systems comprise prokaryotic organisms (e.g., E. coli) and eukaryotic cells (e.g., yeast, HEK293, Xenopus oocytes), which are used to functionally express foreign genes or cDNAs. 

The inhibitors of RNA polymerase, which generates RNA from DNA, inhibit a crucial step in gene expression. Inhibition of the eukaryotic form of RNA polymerase is used in cancer chemotherapy and is also an important experimental tool. For example, actinomy-cin D binds to the guanine residues in DNA and blocks the movement of the eukaryotic RNA polymerase. Specific inhibitors of bacterial RNA polymerase can be used as antibacterial agents. Most of these inhibitors like rifamycin bind to the prokaryotic enzyme. 

Small Ubiquitin-like modifier (SUMO) is a conserved protein that is ubiquitously expressed in eukaryotes and is essential for viability. It serves as a reversible posttranslational modifier by forming an isopeptide bond with lysine residues in many target proteins, in a catalytic process termed SUMOylation. SUMOylation of proteins results in altered inter- or intramolecular interactions of the modified target (Fig. 1). 

The third type of E3 ligases is represented by the polycomb protein 2 (Pc2), which was reported to enhance sumoylation of the substrate CtBP. N- and C-terminal domains in Pc2 that have been implicated in CtBP sumoylation do not resemble known E3 ligases. Like RanBP2, Pc2 expression is restricted to higher eukaryotes. 

Baron U, Schappinger D, Helbl Vet al (1999) Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes. Proc Natl Acad Sci USA 96 1013-1018... 

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