Expression plasmid

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... 

Fig. 4.1.12 Apoaequorin expression plasmid piP-HE (from Inouye et al., 1989, and Shimomura and Inouye, 1999). Reproduced with permission from Elsevier.

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. 

Lu A, Zhang H, Zhang X, Wang H, Hu Q, Shen L, Schaffhausen BS, Hou W, Li L (2004) Attenuation of SARS coronavirus by a short hairpin RNA expression plasmid targeting RNA-dependent RNA polymerase, Virol 324 84-89... 

Fig. 3. Comparison of transfection efficiencies obtained using PolyFect Reagent, a dendrimer-based transfection reagent, and a calcium phosphate-mediated procedure. COS-7 and HeLa cells were transfected in srx-weU plates with a /3-galactosidase expression plasmid using the appropriate protocol. For the calcium phosphate-mediated transfection, 6 pg of plasmid DNA was used and the medium was changed after 5 h incubation. Transfections were performed in triplicate, and transfection efficiency was measured by monitoring the /3-galactosidase activity of extracts obtained from the transfected cells. The amoimt of /3-galactosidase activity in the extracts correlates with the transfection efficiency. Cells were harvested 48 h post-trans-fection...

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... 

Figure 13.5 (A) Schematic representation of expression plasmids used for in vitro tran-...

Technically, in vitro transcription is achieved from standard expression plasmids typically carrying SP6 or T7 promoters using marketed kits. Translation into the polypeptide may be either coupled directly to the transcription (in vitro TnT) or require isolation of the RNA. Again, a large number of suitable prokaryotic and eukaryotic cell extracts as well as complementation factors are freely available. 

PCR reaction using natural dNTPs. However, there are some drawbacks to epPCR. Generally, the technique produces libraries of DNA fragments which need to be ligated into expression plasmids (this can be a limiting step), and some of the methods do not produce random mutations. 

The floxed stop cassette of the integrated construct can be deleted in ES cells by transient transfection with a Cre-expressing plasmid (available, e.g., at http //www.addgene. org/) and subsequent low-density plating as selection for recombined clones is impossible. After electroporation, we plate 500-2,000 cells on feeders in a 10 cm dish and analyze 24 clones for recombination by PCR or Southern blot. At least two recombined clones should be analyzed for knockdown efficiency. 

The activity of the LR Clonase enzymes is so high that considerable degree of dilution of the enzymes is possible. At least, we have succeeded in obtaining proper expression plasmids using 1/40-diluted enzymes (data not shown). 

The specificity and accuracy of the LR reaction seem higher than those of the BP reaction. In addition, there is almost no possibility that some errors (such as mutation and deletion) occur unlike PCR followed by the BP reaction. Therefore, most of the E. coli colony has correct plasmids. However, we recommend to check at least two colonies per expression plasmid to ensure acquisition of proper plasmids. 

Blommel PG, Martin PA, Wrobel RL, Steffen E, Fox BG. (2006) Hi efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system Protein Expr Purif 47, 562-570. 

Fig. 1. Outline of the strategy to construct GST-fused expression plasmids by the in vitro recombination-assisted method. Am, Gm, and Cm are abbreviations for ampicillin-, gentamicin-, and chloramphenicol-resistance, respectively. The figure also indicates the ccdB gene encoding a toxin targeting the co//essential DNA gyrase and the phage X recombination sites (attB, attP, attL, and attR).

PCR and in vitro recombination reactions are quite simple and straightforward for generating multiple expression plasmids in parallel, e.g., in a 96-well plate see Fig. 3a). The first preliminary expression experiment was done to evaluate the production level of each GST-fused protein. In this step, we compared the staining patterns of E. coli proteins harboring expression plasmids with the patterns of proteins harboring empty vectors on sodium dodecyle sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under the same culture conditions see Fig. 3b). In addition to... 

The cells were transfected with 50 ng of HaloTag expression plasmid with 150 ng of pcDNA-DEST47 GFP as a carrier... 

---