Eukaryotic Expression Systems

7 METHODS TO EVALUATE GENE FUNCTION 2.7.1 Eukaryotic Expression Systems 

Numerous eukaryotic expression systems are employed to study gene function in an intact cell, and these most often use a plasmid or viral expression system. In 

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Jebanathirajah, J. A. Andersen, S. Blagoev, B. Roepstorff, P. A rapid screening method to monitor expression of recombinant proteins from various prokaryotic and eukaryotic expression systems using matrix-assisted laser desorption ioniza-tion-time-of-flight mass spectrometry. Anal. Biochem. 2002, 305, 242-250. 

Some disadvantages are common for most eukaryotic expression systems lower growth rates, lower transformation efficiency, and often more expensive cultivation media compared with bacterial systems. 

Higher eukaryotic expression systems are usually used for production of small amounts of protein for example, for analytical purposes or if a native conformation of complex enzymes has to be obtained. They can further be used to modify enzymes which were produced by lower... 

Geisse, S., Gram, H., Kleuser, B., and Kocher, H. P. (1996). Eukaryotic expression systems a comparison. Protein Expr. Purif. 8,271-282. 

The choice of prokaryotic versus eukaryotic expression systems is determined by the functional activity requirement of the protein product. When a therapeutic protein is designed, additional considerations are introduced, including post-translational modifications (i.e., glyco-... 

Most target proteins of therapeutic interest are human or mammalian in origin, but some require post-translational modification during expression for optimum biological and pharmacokinetic properties. Bacterial expression systems do not allow most post-translational modifications. In those cases, eukaryotic expression systems are chosen. These systems, however, are more complex and require more time and resources to engineer, especially in mammalian cells. Consequently, the final products produced in mammalian cells are more expensive than those produced by bacterial expression systems. 

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. 

Expression of eukaryotic genes in yeast has two main advantages (i) the yeast expression system contains many features of a eukaryotic expression system such as glycosylation or disulfide bond formation, and (ii) yeast is a very economical system. Yeasts are single cells, can be cultivated easily, feature fairly short doubling times, and require relatively inexpensive medium ingredients in many aspects they resemble bacteria. 

Various strategies have been used to combine the variable region of antibodies, which bind to the antigen determinants, with small functional proteins. Such constructs can be produced on a large scale in various expression systems (Irving et al., 1996 Roque et al., 2004) bacterial expression systems are relatively simple and less expensive than the alternatives, but eukaryote expression systems (yeast, mammalian, and insect cells) are also being used for this purpose (Roque et al., 2004). 

Geisse S, Gram H, Kleuser B, Kocher HP. Eukaryotic expression systems A comparison. Protein Express Purific 1996 8 271-82. 

B. Eukaryotic Expression Systems 1. Baculovims Expression System... 

Eukaryotic expression systems baculovirus as, 8-10 plant-based, 13-15 vaccinia virus and, 10-12 yeast expression system and, 12-13 European Synchrotron Radiation Facility (ESRF), 40... 

Gellissen G (2008) Production of Recombinant Proteins Novel Microbial and Eukaryotic Expression Systems. Wiley, Hoboken, NJ, USA... 

Over the last two decades, the use of eukaryotic cells for expression of recombinant proteins has become the preferred choice for many applications. This is primarily the case when posttranslational modifications and correct disulfide-bond formation are necessary for protein folding and activity. Among the eukaryotic expression systems, the baculovirus-infected insect cell platform has gained particular attention, resulting in the development and implementation of multiple strategies for protein expression. Here, we present baculovirus-infected insect cells as an efficient expression system for eukaryotic proteins. We demonstrate a simplified and a shortened procedure for recombinant virus production that is sufficient for large-scale production of proteins in insect cells. 

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