Vectors protein expression

The plasmid vectors pBR322 and pUC are referred to as cloning vectors. They are used to insert the foreign DNA and to amplify it. However, if the goal is to produce the protein product from the foreign DNA, they are not suitable. An expression vector is needed. 

The end product, active insulin, has two polypeptide chains as a result. (Adapted with permissionDealing with Genes The Language of Heredity, by Paul Berg and Maxine Singer, 1992 by University Science Books.) 

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FIGURE 13.17 A protein expression vector contains the hybrid promoter derived from fusion of the lac and trp promoters. Expression from pi A more than 10 times greater than expression from either the lac or trp promoter alone. Isopropyl-/3-D-thiogalacto-side, or IPTG, induces expression from pi as well as lac. 

Figure 7.6. Purification of protein from pooled yeast strains. Each yeast ORF was cloned as a fusion to glutathione-S-transferase in a protein expression vector to create 6144 yeast strains. The individual strains were pooled in groups of 96 to create a set of 64 pools. Each pool was grown and the 96 fusion proteins are purified in batch. Each pool was then assayed for a biochemical function (Martzen et al., 1999). Pools positive for function were then deconvoluted using smaller pools consisting of strains from rows and columns of a 96-well plate.

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). 

Plasmid of Ehime University (pEU) protein expression vector. 

The ALA2 BM-3 clone is being sub-cloned into protein expression vectors in attempts to isolate purified P450BM-3 la2 protein for substrate binding... 

Why is it that fusion protein expression vectors contain thrombin and factor Xa recognition sequences rather than trypsin or chymotrypsin recognition sequences Why would it sometimes be desirable to remove the affinity tag from the fusion protein ... 

Fig. 24.6 Expression vectors and the generation of fusion proteins. Expression vectors have optimized all the signals required for transcription (inducible promoter and transcriptional terminator) and for translation (ribosome binding site). Some of them carry the gene for (5-galactosidase with a multiple cloning site that allows the insertion of small genes for the generation of fusion proteins.

Figure 3.16 Typical commercial protein expression vectors, (a) pET-24a (+) the origin of replication (on), kanamycin resistance...

Baculoviruses Protein Expression Vectors and Insect Control Agents... 

Prior to the interest shown in baculoviruses as protein expression vectors, augmentation of natural populations of these insect-specific viruses had been in use for decades, as an insect control strategy (32). Due to the nature of baculovirus infection of lepidopteran larvae, it takes several rounds of viral replication within the larva before the larva succumbs to the infection. This may take firom days to weeks according to environmental conditions. Baculoviruses are used therefore mainly against pests of crops which can sustain some degree of damage, without significant economic loss (32). 

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