C-Terminus pQE Vector Set

This set from QIAGEN provides three vectors — pQE-16, pQE-60, and pQE-70 — for high-level expression of C-terminally 6xHis-tagged proteins in E. coli, and is recommended for open reading frames with "pause sites" that can cause premature termination.

The vectors combine a powerful phage T5 promoter recognized by E. coli RNA polymerase with a double lac operator repression module to provide tightly regulated, high-level expression. Two lac operator sequences increase the probability of lac repressor binding and ensure efficient repression of the T5 promoter. A synthetic ribosomal binding site (RBSII) supports efficient translation, and the 6xHis-tag coding sequence can be placed either 5' or 3' to the polylinker cloning region. Translational stop codons are provided in all reading frames for convenient preparation of expression constructs. Two strong transcriptional terminators — t0 from phage lambda and T1 from the rrnB operon of E. coli — prevent read-through transcription and enhance construct stability. The ColE1 origin of replication is derived from pBR322, and the beta-lactamase gene (bla) confers ampicillin resistance.

pQE-60 and pQE-70 allow the original start codon of the coding fragment to replace the ATG in the vector, preserving the authentic N-terminus of the protein. pQE-16 enables expression of C-terminally 6xHis-tagged DHFR-fusion proteins; DHFR (dihydrofolate reductase) enhances antigenicity and stability and is recommended for poorly expressed proteins or short peptides prone to proteolysis. Because DHFR displays little immunogenicity in mouse and rat, DHFR-fusion proteins are suited for epitope screening. All three reading frames are provided. Expression is performed in vivo in E. coli and is induced by addition of IPTG. In-frame cloning is necessary, no tag removal sequence is included, and the vectors can be maintained in any E. coli strain that is ampicillin-sensitive and carries the pREP4 repressor plasmid or harbors the lacIq gene on the F-factor episome.