pET Trx Fusion System 32
pET vectors plus host strains with induction control
pET Expression Systems and pET
pET Expression Systems and pET Expression Systems plus Competent Cells provide core reagents needed for target gene cloning and expression.
Components: pET Expression Systems
Components for pET Expression Systems are similar for all systems unless otherwise stated with the specific pET Expression System description. pET Expression Systems include:
• 10 µg pET vector DNA (for each indicated plasmid)
• 0.2 ml BL21 Glycerol Stock
• 0.2 ml BL21(DE3) Glycerol Stock
• 0.2 ml BL21(DE3)pLysS Glycerol Stock
• 0.2 ml Induction Control Glycerol Stock
Components: pET Expression Systems plus Competent Cells
pET Expression Systems plus Competent Cells contain all of the components of the specific pET Expression System, as well as the following additional components, unless otherwise stated with the specific pET Expression System description:
• 0.2 ml NovaBlue Competent Cells
• 0.2 ml BL21(DE3) Competent Cells
• 0.2 ml BL21(DE3)pLysS Competent Cells
• 2 × 0.2 ml SOC Medium
• 10 µl Test Plasmid
These components are sufficient for up to 10 transformations in each host.
Purification and Detection Reagents
Purification and detection reagents are available separately. For complete product descriptions and ordering information, refer to the Protein Purification and Antibodies, Conjugates & Detection Tools chapters.
pET Trx Fusion System 32
The popular pET Trx Fusion System 32 is designed for cloning and high-level expression of polypeptide sequences fused with the 109-aa Trx•Tag™ thioredoxin protein. Many proteins that are normally produced in an insoluble form in E. coli tend to become more soluble when fused with the Trx•Tag sequence (LaVallie 1993, Novy 1995). The pET-32 vectors are compatible with the trxB/gor mutant Origami, Origami 2, Origami B, Rosetta-gami, Rosetta-gami 2, and Rosetta-gami B strains. The thioredoxin reductase (trxB) mutation has been shown to allow the formation of disulfide bonds in the E. coli cytoplasm (Derman 1993). The thioredoxin fusion tag expressed from pET-32 vectors not only enhances the solubility of many target proteins, but appears to catalyze the formation of disulfide bonds in the cytoplasm of trxB mutants (Stewart 1998). The glutathione reductase (gor) mutation is the other central enzyme in cytoplasmic disulfide metabolism. Strains containing the trxB/gor mutations promote the highest levels of disulfide bonding in the E. coli cytoplasm (Prinz 1997). Expression of pET-32 constructs in a trxB/gor host may yield the maximum levels of soluble, active, properly folded target protein.
pET-32 is also available in two additional versions prepared for ligation-independent cloning (LIC) of PCR products. With these versions, all vector-encoded N-terminal fusion sequences can be removed from purified proteins with either enterokinase (Ek/LIC) or Factor Xa (Xa/LIC).
Another pET vector with the Trx•Tag thioredoxin protein is pET-48b(+) (Cat. No. 71467-3), which has a human rhinovirus 3C (HRV 3C) protease site for removal of the Trx•Tag fusion.
The pET System allows for expression of target genes under control of the T7 promoter and is covered by US Patent 5,693,489. This patent is licensed by EMD Millipore Corporation from Brookhaven National Labs. Commercial entities need to obtain a license from Brookhaven National Labs prior to use of this Product. Ccommercial use of this Product also requires a commercial license from EMD Millipore Corporation. Commercial use shall include but not be limited to (1) use of the Product or its components in manufacturing; (2) use of the Product or its components to provide a service, information, or data