The pPAL7 vectors are derived from a T7-based expression plasmid and utilize the T7 lac promoter and terminator, and a T7 RNA polymerase expression host for inducible protein production. The plasmids confer ampicillin resistance, constitutively express the lacI repressor for tight control of target gene transcription, and have a pMB1-derived ColE1 origin of replication. Gene targets cloned into Profinity eXact pPAL7 vectors express the prodomain of the subtilisin protease as their fusion partner or tag.
Two pPAL7 expression vector kits are available for the Profinity eXact fusion-tag system – the pPAL7 RIC-ready expression vector kit and the pPAL7 supercoiled expression vector kit. Both contain a 5901-bp pPAL7 vector, but the former contains a versatile predigested form of plasmid for restriction enzyme-free cloning of any target gene, regardless of internal restriction sites, and the latter, a supercoiled plasmid.
Precise fusions in which the resultant purified product does not contain any additional amino acids at the N-terminus may be constructed using the HindIII restriction site and an appropriate restriction site in the multiple cloning site (MCS), or by cloning with the prepared restriction-independent cloning (RIC) vector. Use of the linearized, RIC-ready vector facilitates high-throughput cloning of DNA sequences, regardless of the restriction sites that may be present within a target gene.
Profinity eXact pPAL7 RIC-Ready Expression Vector Kit
The pPAL7 restriction-independent cloning (RIC)-ready expression vector is designed to allow higher throughput and versatility for the cloning of target DNA sequences. The vector is prepared by double digestion of the supercoiled plasmid with SapI and EcoRI, and is then dephosphorylated to reduce postligation background transformants. The target DNA is amplified by PCR using phosphorylated primers containing specific residues at the 5' end that, upon treatment with T4 DNA polymerase, create 5' overhangs complementary to the RIC-ready vector.
Profinity eXact pPAL7 Supercoiled Expression Vector Kit
The pPAL7 supercoiled plasmid expression vector is used for routine cloning of target DNA sequences using a conventional restriction digest cloning strategy. Both the pPAL vector and PCR product are digested with appropriate enzymes found in the multicloning site and ligated.
BL21 (DE3) Chemi-Competent Expression Cells
Chemi-competent cells are also available for cloning and expression of Profinity eXact fusion-tagged proteins. BL21 (DE3) E. coli cells* are the preferred host for T7 vector based protein expression. The cells are DE3 llysogens with the T7 RNA polymerase gene under the control of the lacUV5 promoter. Induction with IPTG allows production of T7 RNA polymerase, which then directs the expression of the target gene located downstream of the T7 lac promoter in the expression vector. The strain is deficient in ompT and lon proteases, which provides improved recombinant protein stability.
* Genotype: E. coli B F– dcm ompT hsdS(rB –, mB –) gal γ(DE3).
Profinity eXact Cloning and Expression Starter Kit
The Profinity eXact cloning and expression starter kit includes everything necessary to clone and express target genes using the Profinity eXact system technology. This comprehensive kit includes both Profinity eXact pPAL supercoiled and RIC-ready expression vectors, chemi-competent cloning cells, BL21 (DE3) chemi-competent expression cells, and SOC growth medium.