Mammalian expression systems

Schaner, M. E., et al. Functional characterization of a human purine-selective, Na+-dependent nucleoside transporter (hSPNTl) in a mammalian expression system. J. Pharmacol. Exp. Ther. 1999, 289, 1487-1491. 

Baculoviruses are members of a large group of double-stranded DNA viruses which only infect invertebrates, including insects. The restricted host range makes baculoviruses safer than mammalian expression systems. The most widely used baculoviruses are Autographa califor-nica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus. The host cell most commonly used is Sf9, derived from the fall army worm Spodoptera frugiperda. 

To date, a single report of a small persistent current in transiently transfected HEK cells is the only evidence for the functional expression of Nay 1.9 in mammalian expression systems [102],... 

Werner RG, Noe W, Kopp K, Schluter M. Appropriate mammalian expression systems for biopharmaceuticals. Arzneimittelforschung 1998 48(8) 870-80. 

Zhang L,GorsetW, Washington CB, BlaschkeTF, Kroetz DE, Giacomini KM. Interactions of HIV protease inhibitors with a human organic cation transporter in a mammalian expression system. Drug Metab Dispos 2000 28 329-334. 

The major advantage of using mammalian cells to produce heterologous eukaryotic proteins are that the expressed proteins are correctly folded and glycosylated to near native structures [ 173]. Because of these unique post-trans-lational modifications, various mammalian expression systems have been reported time to time for the production of therapeutic proteins [173-176]. However, the high cost of cell culture, low productivity (5-50 pg cell 1 day-1), and difficulty in growing in large scale imposes serious restrictions on their... 

Selected mutant VH and VL genes are ready to be transferred to expression construct of choice. Antibodies can be produced in scFv, Fab, or IgG formats depending on biophysical properties of the antibody and the biosensor requirements. The scFv format is perhaps the quickest and easiest to produce in either E. coli or yeast (17) however, stability can be an issue (18). The Fab format is widely used as it is generally more stable than scFv, but expression levels in E. coli can be low and purification more difficult due to the heterodimeric nature of the fragment (19). The IgG format is the most stable and widely used however, the tetrameric quaternary structure dictates mammalian expression system, which can be time consuming and burdensome for the uninitiated (20). 

Fig. 7.4 The HIV-1 lentiviral-based vector contains the 5 long terminal repeat (5 -LTR), transactivation response region (TAR), packaging sequence (0), primer binding site (pbs), splice donor (SD) and acceptor (SA) sites, Rev response element (RRE), the 3 long terminal repeat (3 -LTR) and the polypurine tract (ppt) from HIV-1. It contains a cassette with the tat cDNA placed under the LacS-witch II inducible mammalian expression system...

Today, recombinant protein production involves many options. In addition to E. coli, several yeast systems (see Part IV, Chapter 13), insect cells (see Part IV, Chapter 14), different mammalian expression systems (CHO, BHK, NSO, HKBll, PER.C6) (see Part II, Chapter 3 and Part IV, Chapters 1 and 3) other alternative expression systems are currently under development for the production of biopharmaceuticals. These include transgenic animals or plants, and will be discussed in Part IV, Sub-Part 2 of this book. This chapter will focus on E. coli, a still-modern secretory Saccharomyces ccrevisiac system, and the recently developed mammalian HKBll expression system. An E. coli host/vector system is described that was originally developed for the efficient production of an interleukin-4 variant Later, it transpired that this system is ideally suited to the expression of other proteins and Fab fragments. The secretory... 

The volumetric productivity achievable in simple bioreactor systems when using bacterial expression systems is superior to that of mammalian expression systems. Growth rates are higher, and the ease of scale-up of the fermentation process enables manufacturing at scales up to five-fold larger compared to mammalian systems. 

Verma, R., Boletti, E., George, A.J.T., Antibody engineering comparison of bacterial, yeast, insect and mammalian expression systems. /. Immunol. Methods 199S, 216, 165-181. 

Knorr, T., Nagel, W., and Kolanus, W. (2000). Phosphoinositides determine specificity of the guanine-nucleotide exchange activity of cytohesin-1 for ADP-ribo lation factors derived from a mammalian expression system. Eur. J. Biochem. 267, 3784-3791. 

Drug Metabol. Disposition, 28, 329 (2000). Interactions of HIV Protease Inhibitors with a Human Organic Cation Transporter in a Mammalian Expression System. 

FIGURE 3.2 Ecdysone inducible mammalian expression system. Part I Components of the transfection system. Part 11(a) Uninduced system, (b) Induction of gene expression by addition of inducing agent. 

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