Plasmids gene expression

Use temperature-dependent control of gene expression. Plasmid is more likely to be kept unchanged when the gene expression is repressed. The higher the gene expressions, the more segregants tend to appear. 

Figure 14.2 Basic components of a gene expression plasmid...

LBWIN, B. Gene expression Plasmids and Phages, (197 ) John Wiley and Sons, New York. 

The methods involved in the production of proteins in microbes are those of gene expression. Several plasmids for expression of proteins having affinity tails at the C- or N-terminus of the protein have been developed. These tails are usefiil in the isolation of recombinant proteins. Most of these vectors are commercially available along with the reagents that are necessary for protein purification. A majority of recombinant proteins that have been attempted have been produced in E. Coli (1). In most cases these recombinant proteins formed aggregates resulting in the formation of inclusion bodies. These inclusion bodies must be denatured and refolded to obtain active protein, and the affinity tails are usefiil in the purification of the protein. Some of the methods described herein involve identification of functional domains in proteins (see also Protein engineering). 

The field of DNA vaccination started when eukaryotic expression vectors were injected into the muscle of laboratory animals [2]. The authors observed protein expression for more than 2 months after injection and noted that no special delivery system was required to obtain this expression. Subsequently, it was demonstrated that antibodies can be induced simply by injecting plasmid DNA into the muscle of mice [3]. Subsequent studies found that the injection of expression plasmids also leads to the induction of a cytotoxic T-cell response. After injection, the DNA enters cells of the vaccinated host and the encoded gene becomes expressed. This eventually leads to the induction of a cellular cytotoxic T-cell, T-helper, and/or humoral (antibody) immune response. 

The test system was considerably less sensitive to endosulfan when mouse ER, rather than human ER, was used to mediate (3-gal activity (Ramamoorthy et al. 1997). In similar assays, endosulfan at 10 jM had no effect on (3-gal activity in yeast Saccharomyces) transfected with either the human or rainbow trout ER (Andersen et al. 1999). In addition, no effect was observed on transcriptional activation of HeLa cells transfected with plasmids containing an estrogen receptor as a responsive element (Shelby et al. 1996). Endosulfan also did not induce transient reporter gene expression in MCF-7 human breast cancer cells at an incubation concentration of 2.5 pM (Andersen et al. 1999). Maximum endosulfan-induced ER-mediated luciferase reporter gene expression occurred in vitro in a T47D human breast adenocarcinoma cell line at approximately 10 pM, while 50% expression of luciferase occurred at about 5.9 pM the maximum expression was approximately 59% of the effect from exposure to 0.03 nM estradiol (0.00003 pM) (Legler et al. 1999). Luciferase expression from combined treatment with endosulfan and dieldrin was additive over concentrations ranging from 3 to 8 pM. 

The gene for the dehydratase was expressed in E. coli under lac promoter, and an expression plasmid pOxD 90F was constructed. The transformant was cultivated under optimal condition at 30° C when much of the enzyme was expressed in a soluble form with more than thousand and several hundred times than the wild-type strain per culture (up to more than 50% of the total soluble protein of the... 

Umelo-Njaka, E., Nomellini, J.F., Yim, H. and Smit, J. (2001) Development of small high-copy-number plasmid vectors for gene expression in Caulobacter crescentus. Plasmid, 46 (1), 37 46. 

Nishikawa M, Yamauchi M, Morimoto K, Ishida E, Takakura Y, Hashida M (2000) Hepato-cyte-targeted in vivo gene expression by intravenous injection of plasmid DNA complexed with synthetic multi- functional gene delivery system. Gene Ther 7 548-555... 

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