Gene Transfer and Expression in Mammalian Cells (eBook)

S.C. Makrides (Herausgeber)

eBook Download: EPUB

2003 | 1. Auflage
722 Seiten
Elsevier Reference Monographs (Verlag)
978-0-08-093035-0 (ISBN)

Infos

The production of proteins in mammalian cells is an important tool in numerous scientific and commercial areas. For example, proteins for human therapy, vaccination or diagnostic applications are typically produced in mammalian cells. Gene cloning, protein engineering, biochemical and biophysical characterization of proteins also require the use of gene expression in mammalian cells. Other applications in widespread use involve screening of libraries of chemical compounds in drug discovery, and the development of cell-based biosensors.This book presents a state-of-the-art comprehensive coverage of the technical aspects of gene expression in mammalian cells, written by experienced scientists working at the forefront of the field.

Textauszug

Why choose mammalian cells for protein production?

Savvas C. Makrides1, Holly L. Prentice2

1 EIC Laboratories, Inc., 111 Downey Street, Norwood, MA 02062,USA; Tel.: [phone]; Fax: [phone]

2 Biogen, Inc., 14 Cambridge Center, Cambridge, MA 02142,USA; Tel.: [phone]; Fax: [phone]

E-mail address: [e-mail]

E-mail address: [e-mail]

Abstract

Publisher Summary

There are many different types of hosts to use for production of natural or recombinant proteins: mammalian cells; bacteria, including Gram-negative, Gram-positive, and L-form; filamentous fungi and yeast, including Saccharomyces cerevisiae and Pichia pastoris; insects, including Drosophila melanogaster, Aedes albopictus, Spodoptera frugiperda, and Bombyx mori; Dictyostelium; Xenopus oocytes, and other types of cells, as well as plant tissue culture, transgenic animals and transgenic plants. Progress is continuing in the development of cell-free systems consisting of purified components. The choice of a suitable host cell or expression system for protein production depends on many considerations, such as cell growth characteristics, the ability to effect extracellular expression, post-translational modifications, folding and biological activity of the protein of interest, as well as regulatory and economic issues in the large-scale production of therapeutic proteins. The economics of the selection of a particular expression system requires a cost breakdown in terms of process, design, and other considerations. Key advantages of mammalian cells over other hosts are the ability to carry out proper protein folding, and complex N-linked and authentic O-linked glycosylation of mammalian proteins. Also, mammalian cells posses an extensive post-translational modification machinery, including the ability to produce mature proteins through proteolytic processing.

Abbreviations

• Ad5 adenovirus serotype 5 • DHFR dihydrofolate reductase • EBNA-1 Epstein-Barr virus Nuclear Antigen-1 • EBV Epstein-Barr virus • GS glutamine synthetase • IGF-1 insulin-like growth factor I • MSX methionine sulfoximine • MTX methotrexate • ori origin of replication

There are many different types of hosts to use for production of natural or recombinant proteins: mammalian cells [1] (Table 1); bacteria, including Gram-negative [2], Gram-positive [3,4], and L-form [5,6]; filamentous fungi and yeast, including Saccharomyces cerevisiae and Pichia pastoris [7]; insect, including Drosophila melanogaster, Aedes albopictus, Spodoptera frugiperda, and Bombyx mori [8]; Dictyostelium [9]; Xenopus oocytes [10], and other types of cells, as well as plant tissue culture [11], transgenic animals ([12] and Chapter 24) and transgenic plants [12,13]. In addition, progress continues in the development of cell-free systems consisting of purified components [14,15].

Table 1 Selected mammalian cell lines for gene expression

∗Expression levels listed here are approximations. In general, production yields depend significantly on several factors, including the specific protein under study, gene amplification state, optimization of culture conditions, etc.

The choice of a suitable host cell or expression system for protein production depends on many considerations, such as cell growth characteristics, ability to effect extracellular expression, post-translational modifications, folding and biological activity of the protein of interest, as well as regulatory and economic issues in the large-scale production of therapeutic proteins. The economics of the selection of a particular expression system requires a cost breakdown in terms of process, design, and other considerations [16]. The relative merits of bacterial, yeast, insect, and mammalian expression systems were examined in an earlier review [17]. Since then, expression technology has evolved to meet the range of research and commercial objectives [18]. One of the most exciting developments in the field of gene expression is the use of metabolic engineering to modify biochemical pathways, with the objective of endowing host cells with a spectrum of new properties for robust growth and enhanced protein production (Chapter 15).

Key advantages of mammalian cells over other hosts are the ability to carry out proper protein folding, and complex N-linked and authentic O-linked glycosylation of mammalian proteins. Also, mammalian cells posses an extensive post-translational modification machinery, including the ability to produce “mature” proteins through proteolytic processing.

Protein folding in mammalian cells is achieved during secretion from the endoplasmic reticulum (ER) to Golgi to the extracellular medium. In the ER, oxidized glutathione promotes thiol-disulfide bond exchange, and molecular chaperones also mediate correct disulfide bond formation. Proteins traversing the secretion pathway are protected from intracellular proteases [19], in contrast to bacteria where proteolytic degradation is a major problem [2]. In bacteria, the reducing environment of the cytoplasm inhibits the formation of stable disulfide bonds and disfavors correct folding of complex proteins, often resulting in the formation of inclusion bodies. Although inclusion bodies have advantages, for example, they effectively concentrate the protein, they also require cumbersome solubilization and renaturation procedures that may be inefficient for all but the smallest proteins. A variety of strategies have been used to overcome this limitation [2], including the use of recently developed strains that favor production of proteins with complex cysteine connectivities [20]. These approaches, however, may be ineffective for high-yield bacterial expression of proteins with a large number of disulfide bonds. For example, a genetically engineered soluble form of the human complement receptor type 1 (sCR1) contains 60 disulfide bonds. Although biologically active small fragments of this protein have been produced in bacteria following arduous refolding protocols, to date the full-length sCR1 has been produced only in mammalian cells [21]. Biochemical pathways and their components involved in protein folding and post-translational modifications in mammalian cells are discussed in Chapter 13.

Glycosylation modulates several biochemical and biophysical properties of proteins, including protein folding, secretion, thermostability, antigenicity, catalytic efficiency, recognition and clearance ([22] and Chapter 14). These glycoprotein attributes are particularly important in human therapy where, for example, pharmacokinetic properties [23] and receptor targeting [24] may be dependent on the presence of specific sugars on the protein, or the presence of non-authentic glycosyl derivatives may confer immunogenicity. Although the glycosylation process in prokaryotes exhibits a diversity of glycan compositions and linkage units that rivals that in eukaryotes [25], prokaryotic glycoproteins in general lack the antennae of eukaryotic N-glycans. Some mammalian glycoproteins retain their activity when produced in bacteria. Most, however, must be produced in mammalian hosts to exhibit full, authentic biological activity. Yeast [17] and insect cells [8] do not functionalize proteins with complex oligosaccharides found in mammalian cells, although it is possible to metabolically engineer insect cells for N-glycoprotein sialylation by the insertion of mammalian glycosyltransferase genes [26]. A comparative study of the properties of human interferon-γ produced in Chinese hamster ovary (CHO) cells, the mammary gland of transgenic mice, and baculovirus-infected Sf9 insect cells, demonstrated the significant influence of host cell type on the type of incorporated N-glycans [27].

Other post-translational modifications that may be important for protein functionality require the use of mammalian host cells. A large number of different types of protein modifications has been documented [28,29], including phosphorylation, fatty acid acetylation (palmitoylation, myristoylation, isoprenylation), N-terminal acetylation, C-terminal α-amidation, methylation, and others. Some (most?) of these covalent modifications also occur in Escherichia coli, but they are of minor importance in the large-scale production of recombinant proteins. More important is the ability of mammalian cells to perform proteolytic processing that is necessary for maturation of specific proteins, e.g., insulin, insulin-like growth factor, relaxin, and other proteins. Finally, mammalian cells are preferred for the production of large proteins that require oligomerization of multiple chains, such as antibodies.

Until recently, key disadvantages of mammalian cell culture were its relatively high cost and complicated purification processes necessary for recovery of the secreted recombinant proteins. The high cost of production is mainly due to the use of fetal bovine serum, an expensive medium supplement that also increases the potential risk of virus, prion and mycoplasma contamination (see Chapter 23). The...

Produkt-Details

• Erscheint lt. Verlag: 24.10.2003
• Sprache: englisch
• Themenwelt: Schulbuch / Wörterbuch
• Themenwelt: Informatik ► Weitere Themen ► Bioinformatik
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Zellbiologie
• Themenwelt: Technik
• ISBN-10: 0-08-093035-2 / 0080930352
• ISBN-13: 978-0-08-093035-0 / 9780080930350