Expression transient

P13. Pober, J. S., Bevilacqua, M. P., Mendrick, D. L., Lapierre, L. A., Fiers, W., and Gimbrone, M., Two distinct monokines, interleukin 1 and tumor necrosis factor, each independently induce biosynthesis and transient expression of the same antigen on the surface of cultured human vascular endothelial cells. J. Immunol. 137,1893-1896 (1986). 

Insect cell systems represent multiple advantages compared with mammalian cell cultures (1) they are easier to handle (Table 2.1) (2) cultivation media are usually cheaper (3) they need only minimum safety precautions, as baculovirus is harmless for humans (4) they provide most higher eukaryotic posttranslational modifications and heterologous eukaryotic proteins are usually obtained in their native conformation (5) the baculovirus system is easily scalable to the bioreactor scale. However, because of the viral nature of the system, continuous fermentation for transient expression is not possible - the cells finally die. 

A plasmid-based transient expression system (InsectDirect system from EMD Biosciences Inc., USA www.emdbiosciences.com) will most probably greatly facilitate parallelization and automation for insect cell cultures. It generally gives lower yields, since expression is driven by an early baculoviral promoter, but it is possible to evaluate protein activity and expression level 24 h after transfection. It is also scalable to 1 L volume. The two main disadvantages, namely the large amount of transfection agent required and the limitation in scalability, can probably be overcome in future. 

Edwards, C.P. and Aruffo, A. (1993) Current applications of COS cell based transient expression systems. Current Opinion in Biotechnology, 4 (5), 558-563. 

Schaner, M. E., et al. Transient expression of a purine-selective nucleoside transporter (SPNTint) in a human cell line (HeLa). Pharm. Res. 1997, 14, 1316-1321. 

Fang, X., et al. Functional characterization of a recombinant sodium-dependent nucleoside transporter with selectivity for pyrimidine nucleosides (cNTlrat) by transient expression in cultured mammalian cells. Biochem. 

Excellent protocols for transient expression of fluorescently tagged proteins in N. benthamiana leaves using Agrobacterium-mediated infiltration are available [99, 100],... 

Shirasu, K., Nielsen, K., Piffanelli, P., Oliver, R. and Schulze-Lefert, P. (1999). Cell-autonomous complementation of mloresistance using a bio-listic transient expression system. Plant J. 17, 293-6. 

Agrobacterium-mediated stable transformation -Plant Agrobacterium-mediated stable transformation -Cell culture Transient protoplast transformation Particle bombardment-based transient expression Agrobacterium- mediated transient expression... 

Compared with whole plants, there has been limited development of foreign protein expression systems specifically for use in tissue culture. Some modifications of expression constructs have resulted in improved protein accumulation or have allowed simplified protein recovery. However, in general, modified expression systems have been tested only in a restricted number of cases and have not resulted in the large increases in product yield required for plant cultures to compete with other foreign protein production vehicles. Transient expression techniques, for example using viral vectors, that have been developed for use in whole plants have not yet been applied in plant tissue culture. 

A vector that facilitates high-level protein expression in plant tissue culture, particularly a transient expression system that could be applied to existing wild-type cultures, would be advantageous for in vitro foreign protein production. However, such a system has not yet been developed. The success of this approach depends in part on whether appropriate levels of viral infection, replication and transmission can be established within tissue culture systems. 

Studies have shown that plants can make biologically active recombinant proteins through both transgenic and transient expression approaches. Although the plant post-translational machinery is similar to that of mammalian cells, there are some notable differences, e.g. differences in glycosylation, particularly the absence of sia-lation, which may impact the activity of certain proteins. The absence of mammalian enzymes may prevent complex maturation processes that are critical for the biological activity of proteins such as insulin. Fortunately these shortcomings affect the activity of only a limited number of proteins. 

The P. patens system is being developed by the German biotechnology company Greenovation Biotech GmbH, which is based in Freiburg. The company has developed transient expression systems that allow feasibility studies, and stable production strains that can be scaled up to several thousand liters. 

The tenor of the two documents is similar, although certain aspects are emphasized differently or not addressed at all in one or the other. For example, transient expression systems are not addressed in the EMEA document, but they are in the FDA document.. We will concentrate on the EMEA Points to Consider and refer to the FDA Draft Guidance where appropriate. 

Human carcinoembryonic antigen Mouse/human chimeric IgGl antibody (cT84.66) Antibody - mediated cancer therapy (colon cancer, breast cancer and tumor with epithelial origin) N. tabacum cv petit Havana SRI (Transient expression) 35S MSP + 1 mg/kg FLW 42... 

Seiler-Tuyns A, Walker P, Martinez E, Merillat AM, Givel F, Wahli W (1986) Identification of estrogen-responsive DNA sequences by transient expression experiments in a human breast cancer cell line. Nucleic Acids Res 14 8755... 

Hung, S.I., et al. Transient expression of Yml, a heparin-binding lectin, during developmental hematopoiesis and inflammation, J. Leukoc. Biol., 72, 72, 2002. 

Fig. 3. Evidence for an endogenous Zn2+-binding site in the DAT but not in the NET. (A) Zn2+ inhibition of [3H]dopamine uptake in COS-7 cells transiently expressing hDAT (filled circles) or hNET (open circles). (B) Effect of Zn2+ on binding of the cocaine analog [3H]WIN 35,428 to hDAT (filled circles) and hNET (open circles). Values are percent of control ([3H]dopamine uptake or [3H] WIN 35,428 binding in the absence of Zn2+) expressed as means S.E. of 3-5 experiments performed in triplicate.

Maximal levels of IL-1 /3 mRNA are detected within 1 h exposure to GM-CSF levels then decline to about 50% of maximal by 2 h and then fall to near base-line levels by 8 h (Fig. 7.10). Similarly, levels of IL-1 j8 protein are transiently expressed, broadly following the changes in mRNA levels. Thus, levels of antigenically-detectable protein are maximal by 2-4 h (in cell extracts) and are detected extracellularly in the culture medium by 4 h. Whilst IL-1 (IL-1 a and IL-1/) at 1 ng/ml) and TNF (at 5 ng/ml) are capable of stimulating an increase in IL-1/) mRNA levels, they can synergise with... 

Incubation times of 0.5-1 h are required for detection of mRNA levels (which return to base-line levels by 24 h), and incubation times in excess of 4 h are needed before protein secretion is detected. This transient expression of IL-8 is in contrast to the pattern of production by monocytes, epithelial cells and fibroblasts, which show expression that persists for up to 24 h after stimulation. IL-8 (and related NAP-like peptides) is a powerful neutrophil chemoattractant and, in combination with cytochalasin B, can induce degranulation and activation of the respiratory burst ( 3.5.5). 

In contrast to c-Jun, phosphorylation of the tumor suppressor p53 by CSN-associated kinases targets the protein for degradation by the Ub system [35]. For p53 stability, modification on Thrl55 is most important as shown by mutational analysis [35] and by using different p53 peptides [31]. Mutation of Thrl55 to Val led to stabilization of the transiently expressed p53 mutant in HeLa as well as in HL60 cells [35]. Inhibitors of CSN-associated kinases such as curcumin [18] caused stabilization of cellular p53 followed by massive cell death [35]. 

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