The QIAexpress Type IV Kit gives high expression, up to 50% of total cellular protein.
QIAexpresspQE 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.
Elements present in QIAexpress pQE Vectors
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
t0 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
7. beta-lactamase gene (bla)
Confers ampicillin resistance
Inserts encoding proteins of interest are cloned into appropriate constructs and transformed into a suitable E. colistrain for expression. Expression is induced by addition of IPTG. QIAexpress 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.
The QIAexpress Expression System provides high-level expression of proteins suitable for many applications, including:
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
N- or C-terminal tag
Versatile, complete system for one-step purification
2.5 ml (100 mg/ml; 7000 units/ml, solution)
Unit definition: That amount of enzyme causing the hydrolysis of RNA at a rate such that k (velocity constant) equals unity (Kunitz units) at 25°C and pH 5.0.