Multiple expression vector

In the present protocol, the restriction free (RF) cloning procedure (11,12) is described in detail. RF cloning can be used to introduce the same PCR product into multiple expression vectors harboring identical sequences at the sites of integration. All vectors listed in Fig. 1 are suitable for such parallel cloning. 

These vectors typically have a polylinker adjacent to the SPG promoter. Successive rounds of transcription initiated by SPG RNA polymerase at its promoter lead to the production of multiple RNA copies of any DNA inserted at the polylinker. Before transcription is initiated, the circular expression vector is linearized by a single cleavage at or near the end of the insert so that transcription terminates at a fixed point. 

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... 

Fig. 1. The expression vector pTDI. (a) pTDI vector map. (b) DMA sequence of the pTDI vector around the multiple cloning sites.

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). 

Expression vector with suitable promotor, multiple cloning site, and fusion tag, where applicable (e.g., six-histidine tag). 

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.

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