Qiagen, 32903, C-Terminus pQE Vector Set

25 µg each: pQE-16, pQE-60, pQE-70

Features

- C-terminal 6xHis tag ensures that only full-length protein is purified
- pQE-16 vector for expression of poorly expressed proteins as DHFR fusions
- pQE-16 vector for expression of short peptides as DHFR fusions

Product Details

This set provides 3 vectors (pQE-16, pQE-60, and pQE-70) for expression of C-terminally 6xHis-tagged proteins and is recommended for open reading frames with “pause sites”, which can cause premature termination. pQE-60 and pQE-70 allow the original start codon of the coding fragment to replace the ATG in the pQE vector, preserving the authentic N-terminus of the protein. These two constructs are created by introducing an Nco I and a Sph I restriction site, respectively, at the ATG codon of the insert by PCR or mutagenesis. pQE-16 allows 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. Since DHFR itself displays little immunogenicity in mouse and rat, DHFR-fusion proteins are ideal for epitope screening.

Principle

Element: Description
Optimized promoter/operator element: Consists of the phage T5 promoter and two lac operator sequences, which increase the probability of lac repressor binding and ensure efficient repression of the powerful T5 promoter
Synthetic ribosomal binding site RBSII: For efficient translation
6xHis-tag coding sequence: Either 5' or 3' to the polylinker cloning region
Translational stop codons: In all reading frames for convenient preparation of expression constructs
Two strong transcriptional terminators: t 0 from phage lambda, and T1 from rrnB operon of E. coli , to prevent read-through transcription and ensure stability of the expression construct
ColE1 origin of replication: From pBR322
Beta-lactamase gene ( bla ): Confers ampicillin resistance

QIA express pQE 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 of recombinant proteins in E. coli (see figure " QIA express pQE vector "). Protein synthesis is effectively blocked in the presence of high levels of lac repressor and the stability of cytotoxic constructs is enhanced. The pQE vectors enable placement of the 6xHis tag at either the N- or C-terminus of the recombinant protein.

Procedure

Inserts encoding proteins of interest are cloned into appropriate constructs (for detailed information, see The QIAexpressionist Handbook ) and transformed into a suitable E. coli strain for expression. Expression is induced by addition of IPTG. Vector pQE-TriSystem constructs can be transformed into E. coli , used as a shuttle vector for recombinant protein expression in insect cells, or transfected into mammalian cells.

Applications

- Purification of functional, conformationally active proteins
- Purification under denaturing conditions for antibody production
- Crystallization for determination of three-dimensional structure
- Assays involving protein–protein and protein–DNA interactions

The QIA express Expression system provides high-level expression of proteins suitable for many applications, including: