Heterologous cell systems

Ko, H.J., Park, T.H. (2005) Piezoelectric olfactory biosensor ligand specificity and dose-dependence of an olfactory receptor expressed in a heterologous cell system. Biosens. Bioelectron. 20 1327-1332. 

Wetzel C. H., Behrendt H., Gisselmann G., Storkuhl K. F., Hovemann B. and Hatt H. (2001) Functional expression and characterization of a Drosophila odorant receptor in a heterologous cell system. PNAS 98, 9377-9380. 

Recently, a putative olfactory receptor from Drosophila, Or43a (Clyne et al., 1999 Vosshall et al., 1999), has been expressed in Xenopus laevis oocytes (Wetzel et al., 2001). The receptor expressed in a heterologous cell system was activated by four odorants, i.e. cyclohexanone, cyclohexanol, benzaldehyde, and benzyl alcohol (Wetzel et al., 2001). These experiments not only provided direct evidence for the function of the Or gene, but also demonstrated that the olfactory receptor can be stimulated without an odorant-binding protein. It was demonstrated earlier that PBP was not necessary to obtain pheromone-dependent responses in cultured olfactory receptor neurons of Manduca sexta (Stengl et al., 1992). The possibility that OBPs have been produced in vitro and were present in cultured ORNs could not be excluded. The same argument can not be raised for the heterologous expression of the Drosophila olfactory receptor. While the evidence that Xenopus oocytes responded to odorants in the absence of OBPs does not support the OBP-odorant complex model, it also demonstrated that OBPs are essential for the kinetics of the olfactory system (see below). 

Voltage Clamp Studies on hERG Potassium Channels in Heterologous Cell Systems... 

When transfected in heterologous cell systems, both the D1 and D5 receptors are able to stimulate the production of cAMP (Dearry et al., 1990 Tiberi et al., 1991). Although other signaling pathways have been described, the cAMP pathway stimulated by D1/D5 receptors remains the most extensively studied and, it has been described in a very interesting manner, in detail, at the level of the dorsal and ventral striatum, the main projection areas for mesencephalic dopamine neurons. 

Vosshall LB et al (2000) An olfactory sensory map in the fly brain. Cell 102 147-159 Wanner KW et al (2007) A honeybee odorant receptor for the queen substance 9-oxo-2-decenoic acid. Proc Natl Acad Sci USA 104 14383-14388 Wetzel CH et al (2001) Functional expression and characterization of a Drosophila odorant receptor in a heterologous cell system. Proc Natl Acad Sci USA 98 9377-9380 Wicher D et al (2008) Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels. Nature 452 1007-1011 Wilson RI et al (2004) Transformation of olfactory representations in the Drosophila antennal lobe. Science 303 366-370... 

Heterologous expression systems comprise prokaryotic organisms (e.g., E. coli) and eukaryotic cells (e.g., yeast, HEK293, Xenopus oocytes), which are used to functionally express foreign genes or cDNAs. 

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. 

Although proteins can be expressed in many heterologous production systems, including bacteria such as Proteus mirabilis [1], fungi such as Pichia pastoris [2, 3] and Aspergillus awamori [4] and insect cells [5, 6], the pharmaceutical industry has narrowed down process development to a small number of platform technologies ... 

Heterologous expression systems hERG current (whole cell patch clamp) Mammalian cell expression (CHO, HEK-293, mouse L-ceUs, COS-7) and Xenopus oocytes expression system Witchel et al. 77 Zou et al. 78 Cavero et al. 79 Martin et al. 80 McDonald et al.81... 

The industrial application of CLPs and VLPs is in the development phase. This can be expected since the baculovirus-insect cells system has become one of the most popular systems for heterologous protein and CLP/VLP production at laboratory scale. After defining the appropriate particle composition for the viruses of interest, research is now addressing the engineering issues in this system (Scheme 1). 

The first transient expression system for cytochrome P450 expression was the COS cell system which couples monkey cells with an SV40-based expression vector (Zuber et al., 1986). The cDNA of interest is introduced into a plasmid expression vector under control of a heterologous promoter. The vector is introduced into COS cells via electroporation or another method allowing DNA uptake. The plasmid vector replicates within the cells and cDNA-derived protein accumulates within the cell. 

Human CYPlAl has been expressed in several of the heterologous expression systems including yeast, human lymphoblasts, V79 cells and human fibroblasts. The EROD activity in V79 cells expressing CYPlAl is 50pmol/(mg total protein x min) (Schmalix et al., 1993). In human lymphoblasts, EROD activity of 155 pmol/(mg microsomal protein X min) was obtained (Penman et al., 1994). EROD activity in human fibroblasts expressing CYPlAl cDNA is 1.2pmol/(10 cells x min) or approximately... 

As discussed above, the purification and reconstitution of active PKSs from a variety of heterologous expression systems (including E. coli) are now feasible. Given the substantial tolerance of PKSs toward altered substrates and intermediates, it should therefore be possible to exploit this catalytic potential in a far more powerful way in cell-free systems than in intracellular systems. The primary limitations are with regard to the scale of synthesis. Attempts to stabilize and reuse the enzymes, in conjunction with the development of cheaper sources of natural and unnatural substrates and recycling systems for NADPH, should go a long way toward ameliorating this limitation. 

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