Overexpression

The chiral intermediate (S)-l-(2 -bromo-4 -fluorophenyl) ethanol was prepared by the enantioselective microbial reduction of 2-bromo-4-fiuoroacetophenone [lObj. Organisms from genus Candida, Hansmula, Pichia, Rhodotcnda, Saccharomyces, Sphingomonas, and baker s yeast reduced the ketone to the corresponding alcohol in 

Levodione reductase from C. aquaticum (Overexpressed in E. coli) 

Finally, the last step of the procedure for optimizing experimental conditions is to identify the denaturation temperature of the protein. This step is important because the rotational tumbling rate of a protein increases with temperature, and faster tumbling results in sharper resonance lines. Therefore, the temperature during the NMR experiments should be as high as possible without denaturating the protein. The denaturation temperature can best be determined by either CD-spectroscopy or one-dimensional NMR. 

The selection of the pulse sequence to be used out of the hundreds that have been published depends on the information desired. NMR spectroscopy cannot only be used to determine high-quality three-dimensional structures, but can provide information about the global fold, interactions with other molecules or just the identification of the secondary 

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Many human diseases are caused when certain proteins are either over- or underexpressed. Eor example, breast cancer can be induced by overexpressing certain cellular oncogenes within mammary tissue. To study the disease, researchers produce a line of transgenic mice that synthesize an abnormal amount of the same protein. This leads to symptoms of the disease in mice that are similar to what is found in humans. A protein can be overexpressed by inserting a DNA constmct with a strong promotor. Conversely, underexpression of a protein can be achieved by inserting a DNA constmct that makes antisense RNA. This latter blocks protein synthesis because the antisense RNA binds and inactivates the sense mRNA that codes for the protein. Once a line of mice is developed, treatments are studied in mice before these therapies are appHed to humans. 

Bulk Enzymes. Enzymes such as proteases, amylases, glucose isomerases, and rennin are used in food processing. Similarly proteases and Hpases are used in detergents. CeUulases and xylanases are used in the paper pulp industry. The genes for most of the enzymes used in the various commercial processes have been cloned and overexpressed. Rennin (chymosin) produced from E. coli and A. nigerhas been approved by FDA for use in the dairy industry. 

Antibiotics. The genes involved in the synthesis of a variety of antibiotics have been isolated (34,35). These include antibiotics such as erythromycin, streptomycin, and also peptide antibiotics such as gramicidin and tyrocidin. Characterization of these gene products facUitates the design of novel antibiotics. In addition, overexpression of some of these gene products is also expected to improve the yield of the antibiotic (34,35). 

Another member of the FGF family, FGF-4, protects against radiation-iaduced ceU killing and enhanced the G2 arrest when overexpressed ia a human adrenal cortical carcinoma ceU line (196). This effect is again manifested as the appearance of a shoulder on the survival curve, although neither the control nor the transfected cells undergo radiation-iaduced apoptosis. No differences ia the yield or repair of either SSBs or DSBs have been observed. 

This seems plausible, as the time at which gonocytes become invasive coincides with periods during postnatal life and puberty when Sertoli cells are producing high amounts of hormones. The malignant gonocytes overexpress c-kit so it is possible that they might proliferate in response to stimulation by stem cell factor secreted by the Sertoli cells. ... 

G. J. Opiteck, S. M. Ramirez, J. W. Jorgenson and M. A. Moseley-III, Comprehensive two-dimensional liigh-performance liquid cliromatogr aphy for the isolation of overexpressed proteins andproteome mapping . Awn/. Biochem. 258 349-361 (1998). 

FIGURE 3.10 Constitutive activity due to receptor overexpression visualization through binding and function, (a) Constitutive activity observed as receptor species ([RaG]/[RL0J) and cellular function ([RaG]/ ([RaG] + 3), where P = 0.03. Stimulus-response function ([RaG]/([RaG] + p)) shown in inset. The output of the [RaG] function becomes the input for the response function. Dotted line shows relative amounts of elevated receptor species and functional response at [R]/KG= 1. (b) Effects of an inverse agonist in a system with [R]/ Kq= 1 (see panel a) as observed through receptor binding and cellular function. 

Overexpression of apoaequorin (Inouye et al., 1989, 1991). To produce a large quantity of apoaequorin, an apoaequorin expression plasmid piP-HE containing the signal peptide coding sequence of the outer membrane protein A (ompA) of E. coli (Fig. 4.1.12) was constructed and expressed in E. coli. The expressed apoaequorin was secreted into the periplasmic space of bacterial cells and culture medium. The cleaving of ompA took place during secretion thus the... 

Devine, J. H., et al. (1993). Luciferase from the east European firefly Luciola mingrelica cloning and nucleotide sequence of the cDNA overexpression in Escherichia coli and purification of enzyme. Biochim. Bio-phys. Acta 1173 121-132. 

Inouye, S., et al. (1989). Overexpression and purification of the recombinant calcium-binding protein, apoaequorin./. Biochem. 105 473-477. 

Verhaegen, M., and Christopoulos, T. K. (2002). Recombinant Gaussia luciferase. Overexpression, purification, and analytical application of a bioluminescent reporter for DNA hybridization. Anal. Chem. 74 4378-4385. 

The only commercially available enzyme, FruA from rabbit muscle (class 1), is the most widely investigated, however, it is also the most sensitive under commonly used reaction conditions4. The other types, which can be isolated from overexpressing bacterial sources5-8, typically... 

On pharmacodynamic grounds, tumor resistance may be caused by such diverse mechanisms as the mutation or redundancy of topo II, the overexpression and preferred nuclear localization of proteasome a-type subunits (leading to a anomalous degradation of topo II), genetic deletion or loss-of-function mutations of p53, overexpression of ROS-detoxifying enzymes, overexpression of Bcl-2 (leading to a diminished cyt c release), etc. However, none of these factors would universally predict the development of anthracycline-resistance in a given tumor or another. 

The atherosclerotic plaque overexpresses another chemoattractant, eotaxin, that may mediate mast cell... 

The pro- and antiapoptotic members of the Bcl2 family affect cellular sensitivity to apoptosis and thus to many chemotherapeutic agents [4]. For example, overexpression of the antiapoptotic genes Bcl2 and Bcl-XL in some cell lines and tumors can confer resistance to apoptosis triggered by ionizing radiation. Conversely, overexpression of proapoptotic Bax in experimental tumors can induce apoptosis directly or render such tumors more sensitive to cisplatin and 5-FU. 

The rationale behind CDK inhibition during anticancer treatment is to stop hyperactive cell cycles and to inhibit the activity of cyclins that are frequently overexpressed in human cancer. 

Since COX-2 is overexpressed in tumour cells, such as those of colorectal cancer, it was anticipated that selective COX-2 inhibitors may inhibit tumour growth. 

To study the effect of an increased gene dosage of GR, Reichardt et al. (2000) and van den Brandt et al. (2007) generated rodent models of GR overexpression. These approaches confirmed that GR controls neuronal and immune functions in a dosage-dependent manner. These results highlight the importance of tight control of GR expression in target tissues and may explain... 

Intracellular single-chain mAbs are supposed to prevent membrane localization of the receptors. Antireceptor mAbs conjugated to radionucleides, or to prodrugs, are tested as well, and DNA vaccines may induce an active immune response against RTK overexpressing tumors. 

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