NOVAGEN pET System Manual

Novagen pET System manual iranian biodatacenter

Free Online Download NOVAGEN pET System Manual in PDF Format

The pET System is the most powerful system yet developed for the cloning and expression of recombinant proteins in E. coli . Target genes are cloned in pET plasmids under control of strong bacteriophage T7 transcription and (optionally) translation signals; expression is induced by providing a source of T7 RNA

The pET System is the most powerful system yet developed for the cloning and expression of recombinant proteins in E. coli . Target genes are cloned in pET plasmids under control of strong bacteriophage T7 transcription and (optionally) translation signals; expression is induced by providing a source of T7 RNA polymerase in the host cell. T7 RNA polymerase is so selective and active that, when fully induced, almost all of the cell’s resources are converted to target gene expression; the desired product can comprise more than 50% of the total cell protein a few hours after induction. Although this system is extremely powerful, it is also possible to attenuate the expression level simply by lowering the  concentration of inducer. Decreasing the expression level may enhance the soluble yield of some target proteins. Another important benefit of this system is its ability to maintain target genes transcriptionally silent in the uninduced state. Target genes are initially cloned using hosts that do not contain the T7 RNA polymerase gene, thus eliminating plasmid instability due to the production of proteins potentially toxic to the host cell. Once established in a non-expression host, target protein expression may be initiated either by infecting the host with λ CE6, a phage that carries the T7 RNA polymerase gene under the control of the λp L and p I promoters, or by transferring the plasmid into an expression host  containing a chromosomal copy of the T7 RNA polymerase gene under lac UV5 control. In the second case, expression is induced by the addition of IPTG or lactose to the bacterial culture or using an autoinduction medium. Although in some cases (e.g., with innocuous target proteins) it may be possible to clonedirectly into expression hosts, this approach is not recommended as a general strategy. Two types of T7 promoters and several hosts that differ in their stringency of suppressing basal expression levels are available, providing great flexibility and the ability to optimize the expression of a wide variety of target genes. All of the pET vectors and companion products are available as kits designed for convenient cloning, expression, detection, and purification of target proteins. The pET Expression Systems provide the plasmids and host strains. The background information in Section VII, Ad ditional Guidelines , will help you determine the best vector/host combination for your applicationpolymerase in the host cell. T7 RNA polymerase is so selective and active that, when fully induced, almost all of the cell’s resources are converted to target gene expression; the desired product can comprise more than 50% of the total cell protein a few hours after induction. Although this system is extremely powerful, it is also possible to attenuate the expression level simply by lowering the concentration of inducer. Decreasing the expression level may enhance the soluble yield of some target proteins. Another important benefit of this system is its ability to maintain target genes transcriptionally silent in the uninduced state. Target genes are initially cloned using hosts that do not contain the T7 RNA polymerase gene, thus  eliminating plasmid instability due to the production of proteins potentially toxicto the host cell. Once established in a non-expression host, target protein expression may be initiated either by infecting the host with λ CE6, a phage that carries the T7 RNA polymerase gene under the control of the λ p L and p I promoters, or by transferring the plasmid into an expression host containing a  chromosomal copy of the T7 RNA ely powerful, it is also possible to attenuate the expression level simply by lowering the concentration of inducer. Decreasing polymerase gene under lac UV5 control. In the second case, expression is induced by the addition of IPTG or lactose to the bacterial culture or using an autoinduction medium. Although in some cases (e.g., with innocuous target proteins) it may be possible to clone directly into expression hosts, this approach is not recommended as a general strategy. Two types of T7 promoters and several hosts that differ in their stringency of suppressing basal expression levels are available, providing great flexibility and the ability to optimize the expression of a wide variety of target genes. All of the pET vectors and companion products are available as kits designed for convenient cloning, expression, detection, and purification of target proteins. The pET Expression Systems provide the plasmids and host strains. The background information in Section VII, Additional   Guidelines , will help you determine the best vector/host combination for your application

Spread the love

Leave a Reply

Your email address will not be published. Required fields are marked *