QIAexpress Expression System

The QIAexpress Expression System is designed for high-level expression of His-tagged proteins. QIAexpress pQE vectors are available to place the small 6xHis-tag coding sequence at the 5 prime or 3 prime end of the construct. QIAexpress Kits are complete kits for efficient expression and one-step purification of 6xHis-tagged proteins. Several pQE-based vectors have been developed that contain additional features for special applications. In addition to a 6xHis tag, the pQE-100 DoubleTag Vector encodes Tag·100, a second epitope for detection of proteins immobilized via the 6xHis tag — especially useful for improved assay design. The new pQE-TriSystem Vector allows parallel expression of His-tagged proteins in E. coli, baculovirus-infected insect cells, and mammalian cells, using a single construct. TAGZyme pQE vectors are used in combination with TAGZyme Enzymes for the exoproteolytic cleavage of His tags from the N-termini of recombinant proteins. The pQE-30 Xa Vector introduces a Factor Xa Protease recognition site into a protein allowing efficient endoproteolytic cleavage of the His tag. Vector pQE-30 UA allows direct insertion of PCR products into an expression vector, without the need for restriction enzyme digestion of vector or insert.

* Encodes RGS·His epitope

Features and benefits

• High-level prokaryotic expression — up to 50% of total cellular protein
• Tightly regulated expression — enhanced stability of cytotoxic constructs
• Flexible cloning — multiple cloning options to place 6xHis tag at N- or C-terminus
• Versatile, complete system — 6xHis tag allows one-step purification, sensitive detection, and improved assay procedures

Principle

QIAexpress 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 table "Elements present in QIAexpress pQE Vectors",and figure "pQE Vectors") enable placement of the 6xHis tag at either the N- or C-terminus of the recombinant protein.

QIAexpress vectors provide different options for different cloning needs. Vectors that place the 6xHis tag at the N-terminus are the most commonly used, and as they only need to be cloned in-frame at the 5' end of the insert, they are also the easiest to use. Vectors that place the 6xHis tag at the C-terminus are recommended for open reading frames with “pause sites” that cause premature translation termination, to ensure that only full-length proteins are purified. pQE-16 and pQE-40 allow fusion of short peptide-encoding sequences to the murine dihydrofolate reductase (DHFR) sequence with a C- or N-terminal 6xHis tag respectively. This enhances both stability and antigenicity of the peptide, making DHFR fusions ideal for antibody production.

pQE Vectors

pQE vectors. For a description of numbered features, see table "Elements present in QIAexpress pQE Vectors". Diagram not to scale.

Elements present in QIAexpress pQE Vectors

The vectors in the pQE-80L series (cis-Repressed Vector Set) express the laclq gene product that represses protein expression prior to IPTG (isopropyl-beta-D-thiogalactopyranoside) induction. These vectors simplify the characterization of plasmids during vector construction and make it easier to use any E. coli strain for protein expression by eliminating the need to include a second plasmid that encodes the repressor in trans (e.g., pREP4, see below).

Any E. coli strain carrying the laclq mutation can be used for expression of most proteins with pQE vectors. For stable propagation of expression constructs encoding ”toxic” or hydrophobic proteins, a higher level of lac repressor may be required. This is obtained in trans with repressor plasmid pREP4 or in cis with the pQE-80L vector series, both of which constitutively express the lac repressor at high levels. Expression hosts M15[pREP4] and SG13009[pREP4] are provided in QIAexpress Kits and in the E. coli Host Strains Set. Alternatively, pREP4 can be transformed into any E. coli strain. Since pREP4 contains a p15A origin of replication, it is compatible with plasmids containing a ColE1 origin of replication, such as the pQE vectors, and is maintained in the cell by kanamycin selection.

New UA-cloning technology allows the direct insertion of PCR products generated using Taq or other non-proofreading DNA polymerases into the pre-linearized pQE-30 UA expression vector included in the QIAexpress UA Cloning Kit.

The pQE-100 DoubleTag Vector allows proteins to be tagged at the N-terminus with the 6xHis tag and at the C-terminus with the Tag·100. The vector has been specifically designed to enable assays that do not require antibodies specific for each individual protein of interest. Proteins are immobilized via their 6xHis tags to Ni-NTA HisSorb Strips or Plates, or Ni-NTA Magnetic Agarose Beads, and are then detected using the Tag·100 Antibody, which specifically recognizes the Tag·100 epitope. This allows a wide range of assays to be designed.

The pQE-TriSystem Vector contains T5, p10, and CAG promoters that enable His-tagged protein expression in E. coli, baculovirus-infected insect cells, and mammalian cells respectively.

Since it is seldom necessary to remove the small 6xHis tag after purification, most QIAexpress pQE vectors do not encode a protease cleavage site. However, for some applications, affinity tag removal is desirable. QIAGEN offers two systems for efficient affinity-tag removal. The TAGZyme System is based on highly specific exoproteolytic digestion, and the Factor Xa Protease System utilizes the endoprotease Factor Xa Protease.

The TAGZyme System employs three recombinant enzymes for the highly specific and efficient exoproteolytic removal of vector-encoded sequences from the N-termini of recombinant proteins expressed using TAGZyme pQE vectors.

The pQE-30 Xa Vector encodes a Factor Xa Protease recognition site between the 6xHis tag and the body of the protein, which allows efficient endoproteolytic cleavage of vector-encoded amino acids using Factor Xa Protease.

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. 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

The QIAexpress Expression System provides high-level expression of proteins suitable for
many applications, including:

• Purification of functional, conformationally active proteins (1)
• Purification under denaturing conditions for antibody production (2)
• Crystallization for determination of three-dimensional structure (3)
• Assays involving protein–protein (4) and protein–DNA interactions

Handbook

All QIAexpress vectors are supplied with a comprehensive handbook,  The QIAexpressionist.

Cited References

1. Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K. Jr. (1997) Direct observation of the rotation of F1-ATPase. Nature 386, 299.
2. Gu, J., Stephenson, C.G., and Iadarola, M.J. (1994) Recombinant proteins attached to a nickel-NTA column: use in affinity purification of antibodies. BioTechniques 17, 257.
3. Flachmann, R. and Kühlbrandt, W. (1996) Crystallization and identification of an assembly defect of recombinant antenna complexes produced in transgenic tobacco plants. Proc. Natl. Acad. Sci. USA 93, 14, 966.
4. Greiner, S., Krausgill, S., and Rausch, T. (1998) Cloning of a tobacco apoplasmic invertase inhibitor. Plant Physiol. 116, 733.

For more references using the QIAexpress System, visit the QIAGEN Reference Database online .