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pET Express & Purify Kit—HisTALON Bacterial Expression & Purification—pET Express & Purify Kits
The pET expression system is the most commonly used bacterial system for the over-expression of genes. We offer a complete system with a choice of N- or C-terminal 6xHN-tagged vectors together with IMAC-based purification.The pET Expression System Provides the Highest Level of Protein Expression The pET system utilizes two levels of regulation to provide the highest level of protein expression and the tightest control over basal expression—achieved via the presence of lac operator sites in two different promoters.
The first level of regulation is provided by the pET6xHN series of vectors. The gene of interest is cloned downstream of a strong T7 lac hybrid promoter, which combines the T7 promoter with the lac operator. T7 RNA polymerase is extremely selective in binding to this hybrid promoter, thereby utilizing most of the cell's resources to express this gene.
•The second level of regulation occurs in the host cell. The T7 RNA polymerase gene is integrated into the host genome under the control of the lac UV5 promoter, which also contains a lac operator. This enables expression of T7 RNA polymerase to be controlled by the lacI genes present in both the host genome and the pET6xHN vectors, which encode lac repressor. In the uninduced state lac repressor inhibits expression of both T7 RNA polymerase and the gene of interest. When IPTG is added during induction, it binds to lac repressor, which then dissociates from the lac operators, removing this inhibition. This allows expression of T7 RNA polymerase, which in turn binds to the newly derepressed T7 lac hybrid promoter and transcribes the gene of interest. The RNA transcript is then translated, leading to a very high level of target protein expression within the host cell.
The pET Express & Purify System Vectors—pET6xHN Clontech’s pET Express and Purify kits contain pET vectors (the pET6XHN series of vectors) which encode N- or C-terminal 6xHN fusion tags. These are available in a choice of cloning formats (easy In-Fusion cloning or traditional restriction enzyme cloning) for maximum flexibility tailored to your expression needs. pET Express & Purify Kits—a Complete Expression & Purification System
The pET Express & Purify kits supply a choice of IMAC resins and buffers to purify expressed his-tagged proteins. You can choose His60 Ni nickel-based resin for high binding capacity or HisTALON cobalt-based resin for high purity. The kits are supplied with prepacked gravity columns filled with either resin and provide all of the buffers necessary to perform the protein extraction and purification.Cobalt-Based Resin—HisTALON
No copurification of unwanted host proteins No SlyD contamination (histidine-rich protein present in E. coli) Lowest metal ion leakage Available in various formats as bulkresin, cartridges, and gravity columns Nickel-Based Resin - His60 Ni Superflow Resin
60 mg/ml binding capacity Up to 95% purity Low metal ion leakage Available in various formats as bulk resin, cartridges, and gravity columns |