Bacterial expression vectors

RNA to initiate cDNA synthesis. All cellular mRNA contains multiple repeats of adenine bases (poly-A tails). Therefore the complementary thymine bases (oligo-dT) can be used as a primer that binds to the mRNA template required for the reverse transcriptase to synthesize the cDNA. In the case of pancreatic mRNAs (Figure 4.2), the signihcantly higher mRNA for insulin compared with other proteins allowed success in isolating the insulin-specihc cDNA. Subsequent insertion of cDNA into a bacterial expression vector allowed the production of functional insulin that is now marketed as a successful therapeutic product (Figure 4.2). 

The ior-CEAl-(scFv)2 was supplied by the Centre for Genetic Engineering and Biotechnology (Havana, Cuba). The inducible bacterial expression vector pACR-1 was used to synthesize the diabody in the periplasm of Escherichia coli, according to the conditions described in Ref. [4.7]. Briefly, the genes encoded for the and V, regions of the anti-CEA specific mouse... 

Bacterial expression vectors developed for protein footprinting... 

The ELIP specific coding region of a cDNA-clone was cloned into the bacterial expression vector pEXl (5, 6). Antibodies were raised against the fusionprotein expressed by this expression clone. 

In t ie following section, we will illustrate the in silico creation of a genetic design construct. First, we will construct a bacterial expression vector from basic building block components. We will show how use of hierarchical groups simplifies the reuse of sequence elements to create a second vector for expression of proteins in mammalian systems. We will then show how to create a polypeptide-encoding element, clone it into the vector, and backtranslate for optimal expression in a specified host. 

Some of the elements and groups from the bacterial expression vector can be reused to create an expression vector for use in... 

The cloning tool in Gene Designer is launched from the Tools menu in the Project Window (Fig. 8). The bacterial expression vector is first dragged into the Input Pane to serve as a fragment donor. 

Huang etal. (2002) prepared an antibody array for the simultaneous detection of 43 cytokines. They were able to verify the down-regulation of MCP-1 cytokine in transfected cells (human glioblastoma cells transfected with cx43 expression vector) relative to control cells. The antibody array is an emerging technology. In at least one study based upon the use of a commercial membrane format, the cytokine microarray failed to accurately determine cytokine levels in bacterial and lipopolysaccharide (LPS)-stimu-lated whole human blood (Copeland, 2004). 

There exist a variety of vectors for cloning into eukaryotic systems, ranging from yeast (Saccharomyces as well as Pichia) through insect cells (Baculovims) and plants (Ti plasmid from Agrobacterium tumefaciens) to mammalian cells (transfected by viral or mammalian vectors). As expression in eukaryotic hosts is less efficient than bacterial expression in terms of yield and time and more complicated in terms of vector structure and culture conditions, such eukaryotic expression systems are only used for genes whose proteins require posttranslational modification which is not possible in bacteria. Yeast is the preferred option as a relatively easily culturable single-cell system but posttranslational modification capabilities is limited. The additional complexity can be circumvented in part by exploiting the ability of eukaryotic vectors to act as shuttle vectors, which can be shuttled between two evolutionarily different hosts. Thus, eukaryotic vectors can be replicated and analyzed in bacteria and transfected into eukaryotic cells for expression of the recombinant product. 

The targeting of antibodies to the periplasm requires the use of signal peptides. The pelB leader of the pectate lyase gene of Erwinia carotovora (56) is commonly used. The gill leader (9), the phoA leader of the E. coli alkaline phosphatase, and the ompA leader of E. coli outer membrane protein OmpA have also been used, being common to many protein expression vectors (57,58). Further examples are the heat-stable enterotoxin II (stll) signal sequence (47) and the bacterial chloramphenicol acetyltransferase (cat) leader (59). 

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