Expression in Eukaryotic Systems

Taniguchi, N. Mantel, S. Schwaizstein, S. Nagata, M. Muramatsu, and C. Weissmann, Nature (London), 1980,285,547. 

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Expression of recombinant proteins in eukaryotic systems is a laborions, time-consuming, and costly process. However, despite this unavoidable complexity, expression in eukaryotic systems has gained increasing popularity within the scientific community over the last two decades. The main reason for this is that other expression systems, and primarily the bacterial Escherichia coli system, cannot properly express many proteins, primarily those requiring posttranslational modifications and/or formation of di-sulfide bonds for proper fimction. 

Volume 306. Expression of Recombinant Genes in Eukaryotic Systems Edited by Joseph C. Glorioso and Martin C. Schmidt... 

Post-translational modifications, such as phosphorylation, complex glycosylation, and lipidation, typically occur in eukaryotic organisms. Therefore, their expression in prokaryotic systems like Escherichia coli is difficult. However, it should be noted that via clever engineering and coexpression of specific enzymes, access can be granted to specific lipidated proteins via expression in bacteria, for example, via the expression of A -myristoyltransferase in E. coli Eukaryotic systems that can be used for the expression of post-translationally modified proteins are yeast and Dictyostelium discoidum. Furthermore, lipidated proteins, such as the Rah proteins, can be obtained via purification from tissue sources or from membrane fractions of insect cells that had been infected with baculovirus bearing a Rah gene. ... 

There exist a variety of vectors for cloning into eukaryotic systems, ranging from yeast (Saccharomyces as well as Pichia) through insect cells (Baculovims) and plants (Ti plasmid from Agrobacterium tumefaciens) to mammalian cells (transfected by viral or mammalian vectors). As expression in eukaryotic hosts is less efficient than bacterial expression in terms of yield and time and more complicated in terms of vector structure and culture conditions, such eukaryotic expression systems are only used for genes whose proteins require posttranslational modification which is not possible in bacteria. Yeast is the preferred option as a relatively easily culturable single-cell system but posttranslational modification capabilities is limited. The additional complexity can be circumvented in part by exploiting the ability of eukaryotic vectors to act as shuttle vectors, which can be shuttled between two evolutionarily different hosts. Thus, eukaryotic vectors can be replicated and analyzed in bacteria and transfected into eukaryotic cells for expression of the recombinant product. 

Regulationof gene expression in eukaryotes proceeds primarily by control of transcription as in prokaryotes. Some systems are also regulated at the translational level. 

Although the nature of the bacterial cell makes possible specific control over transcription in ways that cannot be achieved in eukaryotic systems, control of gene expression at the level of translation is also important in bacteria. Control of translation is achieved primarily by the prevention of ribosomal binding. 

In the first gene fusions with GUS as a reporter enzyme the control sequences from the coli lac operon directed GUS expression in coll (17). These experiments were followed by the system s first implementation in eukaryotic systems. Chimeric... 

Volume 306. Expression of Recombinant Genes in Eukaryotic Systems... 

The enhanced expression of metal-induced stress proteins is controlled primarily at the transcriptional level similar to the induction of hsps by heat (Wu et al. 1986). Regulation of hsp genes in eukaryotic systems is mediated by a cw-acting heat shock control element (HSE) that is found in multiple copies upstream of the transcriptional start site (Pelham 1982). Transcriptional activation of the hsp genes is mediated by a ran -acting protein, known as the heat shock factor (HSF), which binds specifically to the HSE (Wu 1984a,b). 

Baron U, Bujard H. Tet repressor-based system for regulated gene expression in eukaryotic cells principles and advances. Methods Enz3miol 2000 327 401-421. 

Human APases (see further below) have also been cloned and molecularly well characterized. Like CIAP, thermolabile human intestinal APase (HIAP) may thus find general uses, particularly in the area of fusion gene expressions in eukaryotic cells. Human placental APase (10) and rat bone/liver/kidney APase (11) have been used as reporters in APase fusion expression systems. When combined with chemiluminescence technology, these enzymes offer a simple and convenient detection system which is at least as sensitive as the conventional CAT detection system. 

Type I CAT (or CAT]), which is typically encoded by transposon Tn9, has been widely used both as a selection marker and as a reporter for studying gene expression in eukaryotic cells. The CAT reporter system is a powerful and sensitive tool for such studies since eukaryotic cells frequently used in such experiments do not have endogenous CAT or CAT-like activities. Furthermore, CAT activity can be detected and quantitated with relative ease and at high sensitivities. 

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