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Ebook Analysis of genes and genomes: Part 2

Part 2 book “Analysis of genes and genomes” has contents: Protein production and purification, genome sequencing projects, post-genome analysis, engineering plants, engineering animal cells, engineering animals. | 8 Protein production and purification Key concepts Proteins are over-produced by placing the gene encoding them under the control of a strong promoter Strong, inducible promoters allow the production of toxic proteins and for proteins to be made under defined conditions Proteins may be produced in bacterial or eukaryotic cells DNA encoding a protein purification tag is often added to the expressed gene to aid in the protein purification process Protein purification tags impart a unique property to the overproduced protein such that it may be purified biochemically The production and purification of proteins for biochemical and structural analysis have formed the lynchpin of many advances in genetic engineering, drug discovery and medicinal chemistry over recent years. Some proteins are naturally expressed at high levels. For example, actin and certain heat-shock proteins can accumulate at high levels within cells. Many other, potentially biologically important, proteins are expressed at very low levels. For example, many transcription factors involved in turning sets of genes on and off are present at only a few copies per cell. To aid the study of proteins that are produced at a low level, the gene encoding them generally has to be overexpressed. The most straightforward way to achieve this is to fuse the target gene to a strong promoter. The strong promoter, usually derived from a highly expressed gene, will drive the expression of any gene placed under its control through the recruitment of RNA polymerase to that gene. Much work has gone into the design of vectors for maximizing protein production. The architecture of a typical expression vector is shown in Figure 8.1. Analysis of Genes and Genomes Richard J. Reece 2004 John Wiley & Sons, Ltd ISBNs: 0-470-84379-9 (HB); 0-470-84380-2 (PB) 258 PROTEIN PRODUCTION AND PURIFICATION 8 Multiple cloning site Pr om r ote Transcriptional terminator RBS Expression vector Selectable marker Origin .