Qiagen, 32149, QIAexpress Type IV Kit

5 µg each: pQE-30, pQE-31, pQE-32 (N-terminal 6xHis); 10 ml Ni-NTA Agarose

Features

- Vectors covering all three reading frames
- Complete kit for expression and purification of His-tagged proteins
- High-level expression of N-terminally His-tagged proteins
- Versatile, complete system for one-step purification and sensitive detection

Principle

Element: Description
1. 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
2. Synthetic ribosomal binding site RBSII: For efficient translation
3. His-tag coding sequence: Either 5' or 3' to the polylinker cloning region
4. Translational stop codons: In all reading frames for convenient preparation of expression constructs
5. Two strong transcriptional terminators: t 0 from phage lambda, and T1 from the rrnB operon of E. coli , to prevent read-through transcription and ensure stability of the expression construct
6. ColE1 origin of replication: From pBR322
7. 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 . 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 (see figure pQE Vectors and table) enable placement of the His tag at either the N- or C-terminus of the recombinant protein.

Procedure

Inserts encoding proteins of interest are cloned into appropriate constructs and transformed into a suitable E. coli strain for expression. Expression is induced by addition of IPTG. QIA express Type IV Kit 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: