Ebook Analysis of genes and genomes: Part 2
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Ebook Analysis of genes and genomes: Part 2
8 Protein production and purificationKey concepts♦Proteins are over-produced by placing the gene encoding them under the control of a strong promoter♦ Ebook Analysis of genes and genomes: Part 2♦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♦P Ebook Analysis of genes and genomes: Part 2rotein purification tags impart a unique property to the overproduced protein such that it may be purified biochemicallyThe production and purificatioEbook Analysis of genes and genomes: Part 2
n of proteins for biochemical and structural analysis have formed rhe lynchpili of many advances in generic engineering, drug discovery and medicinal 8 Protein production and purificationKey concepts♦Proteins are over-produced by placing the gene encoding them under the control of a strong promoter♦ Ebook Analysis of genes and genomes: Part 2at high levels within cells. Many other, potentially biologically important, proreins are expressed ar very low levels. For example, many transcription factors involved in turning sets of genes on and off arc present ar only a few copies per cell. I o aid rhe study of proreins that are produced at a Ebook Analysis of genes and genomes: Part 2 low level, the gene encoding them generally has to he overexpressed. I he most straightforward way ro achieve this is ro fuse rhe target gene to a stEbook Analysis of genes and genomes: Part 2
rong promoter. The strong promoter, usually derived from a highly expressed gene, will drive rhe expression of any gene placed under its control throu8 Protein production and purificationKey concepts♦Proteins are over-produced by placing the gene encoding them under the control of a strong promoter♦ Ebook Analysis of genes and genomes: Part 2 a typical expression vector is shown in Figure 8.1.Anatyiis of Gems and Gtnomes Richard J. R«cce 2004 John Wiley & Sons, Ltd ISBNs: 0-470-84.Ỉ79-9 ÍHBI; 0-470.84)80-2Ebook Analysis of genes and genomes: Part 2
ibosome binding site (RBS) is included to promote efficient translationSuch vectors will often contain a multiple cloning sire located between a stron8 Protein production and purificationKey concepts♦Proteins are over-produced by placing the gene encoding them under the control of a strong promoter♦ Ebook Analysis of genes and genomes: Part 2d a selectable marker such that rhe vector may be autonomously replicated and maintained within cells.Ar high levels, many proteins will be toxic to rhe host cell in which they are produced. Indeed, some proteins when produced in small amounts will also be toxic to rhe host. For example, rhe express Ebook Analysis of genes and genomes: Part 2ion of rhe poliovirus 3AB gene product is highly toxic to E. colt cells, due to the drastic changes it creates in rhe membrane permeability of rhe bacEbook Analysis of genes and genomes: Part 2
teria (Lama and Carrasco, 1992). Therefore, to maximize protein expression it is vital that an inducible expression system be established, so large qu8 Protein production and purificationKey concepts♦Proteins are over-produced by placing the gene encoding them under the control of a strong promoter♦ Ebook Analysis of genes and genomes: Part 2 rhe cells harvested soon afterwards prior to the potentially toxic effects of the expressed protein. Here, we will discuss a number of inducible expression systems that are in common use today. Additionally, we will describe the common host-vector systems that are used for protein production in E. Ebook Analysis of genes and genomes: Part 2colt, yeast, insect and mammalian cells.8.1 Expression in E. collE. colt remains the host cell of choice for the majority of protein expression experiEbook Analysis of genes and genomes: Part 2
ments. Its rapid doubling rime (approximately 30 min) in simple defined8.1 EXPRESSION IN E. coli 259(and inexpensive) media, combined with an extensiv8 Protein production and purificationKey concepts♦Proteins are over-produced by placing the gene encoding them under the control of a strong promoter♦ Ebook Analysis of genes and genomes: Part 2ganism. Additionally, E. coli cells arc easily broken for the harvesting of the proteins produced within the cell. Of course, E. colt does suffer from rhe fact that is a prokaryotic organism when it is used to produce eukaryotic proteins. E. coli cells are unable to process introns and do not posses Ebook Analysis of genes and genomes: Part 2s the extensive post-translational machinery found in eukaryotic cells that can glycolylare, methylate, phosphorylate or alter rhe initially producedEbook Analysis of genes and genomes: Part 2
protein in other ways, such as through extensive disulphide bond formation, The use of cDNA ro produce an expression vector overcomes the first of the8 Protein production and purificationKey concepts♦Proteins are over-produced by placing the gene encoding them under the control of a strong promoter♦ Ebook Analysis of genes and genomes: Part 2ifferent promoter sequences have been used ro illicit inducible protein production in E. coli (Mak rides, 1996). Some of these arc discussed below.8.1.1The lac PromoterWe have already seen (Figure 1.23) that the E. coli lac promoter provides a mechanism for inducible gene expression. The lac genes a Ebook Analysis of genes and genomes: Part 2rc expressed maximally when E. coif are grown on lactose. Fusing the lac promoter sequences to another gene will result in the lactose- (or ĨPTG-) depEbook Analysis of genes and genomes: Part 2
endent expression of that gene, rhe lac promoter suffers, however, from a number of problems that mean that it is rarely used to drive the expression 8 Protein production and purificationKey concepts♦Proteins are over-produced by placing the gene encoding them under the control of a strong promoter♦ Ebook Analysis of genes and genomes: Part 2e transcribed to a significant level in rhe absence of induction (Groncnborn, 1976). The latter problem can be partially overcome by expressing mutant versions of rhe lad gene that have increased DMA binding (and consequently repressing) ability - for example the lacT1 allele results in the overprod Ebook Analysis of genes and genomes: Part 2uction of EđCĨ and consequently results in a reduced level of transcription in rhe absence of inducer (Miiller-Hill, Crapo and Gilbert, 1968).8.1.2TheEbook Analysis of genes and genomes: Part 2
tac Promoterrhe ease with which rhe lac promoter can be activated (rhe addition of IPTG to E. colt cultures) makes it an attractive system for produc8 Protein production and purificationKey concepts♦Proteins are over-produced by placing the gene encoding them under the control of a strong promoter♦ Ebook Analysis of genes and genomes: Part 2 of many E. coli promoters, consensus sequences for the —35 and —10 regions, to which rhe RNA polymerase must bind to transcribe rhe gene, can be determined (I.isser and Margalit, 1993). The lac promoter is weak because the —35 region deviates from rhe consensus (Figure 8.2). The creation of a fusio Ebook Analysis of genes and genomes: Part 2n sequence260 PROTEIN PRODUCTION AND PURIFICATION 8-35-10consensusconsensusS'-TT«ACA-3’S’-TA7AAr-3‘lac: 5-■ .TrrACACTrTA7u::.-:c.:U0CrcyfAfAfl-01-0TaGEbook Analysis of genes and genomes: Part 2
AArfyrttAtZ»aATAACAATTTCACACA0GAAACA .35-10RBSProtein"trp t S’-.. .|ĩTGACAA7TAATCAĨCaAAr7AGĨTAACTAGTACGCAAG7rCACỬĨ?lAAAAGGG7ATCGACA ATG...-J•-35-10RBS8 Protein production and purificationKey concepts♦Proteins are over-produced by placing the gene encoding them under the control of a strong promoter♦8 Protein production and purificationKey concepts♦Proteins are over-produced by placing the gene encoding them under the control of a strong promoter♦Gọi ngay
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