The QIAexpressionist™ Kit, A System for the Expression and Purification of Recombinant Proteins From Qiagen

Qiagen’s protein expression and affinity purification system uses the short, 6 Histidine sequence at the N-terminus of heterologous proteins as an affinity tag for purifying recombinant proteins from crude cell lysates with a single chromatography step with Ni-NTA affinity resin (nickel chelating resin). The His₆ tag purification system is excellent because of its efficient binding and elution characteristics. Another great advantage of this system is the small size of the His₆ affinity tag. In comparison to other larger affinity tags, the His₆ tag is less likely to affect the functional activity of the protein of interest. Furthermore, 6xHis tag is not immunogenic, and at pH8.0 the tag is uncharged, and therefore does not generally affect secretion, folding and compartmentalization. Another use of this tag is that it allows immobilization of the fusion protein to metal chelating surfaces such as Ni-NTA HisSorb™ and therefore, simplifies protein-protein interaction studies. Lastly, an anti-His₆ antibody can be used for detection.

The plasmid

The pQE30 vectors in this kit include pQE30-32. They belong to the pDS family and were derived from pDS56/RBSII. High level protein expression is based on the T5 powerful promoter, which has been optimized with an operator element consisting of two lac operator sequences that increase lac repressor binding and ensure tight repression of the T5 promoter. All three plasmids (3.4 kb) contain, in order, the 5’ end of the T5 promoter, lacO, synthetic ribosome-binding site (RBSII), ATG start codon, 6xHis tag sequence, multiple cloning site (MCS) and translational stop codons in all reading frames for convenient subcloning, Col E1 origin of replication, and â-lactamase for ampicillin resistance at 100 µg/mL. The lacI^q repressor gene is expressed from a separate plasmid, pREP4, already transformed into the M15 or SG13009 E. coli cells. The difference between the three vectors is the linker length between the His₆ tag and the MCS, which provides cloning in all frames to the His₆ tag. After the sequence encoding the protein is cloned into one of the pQE30-32 vectors, protein expression is induced by the addition of isopropyl-thio-D-galactoside (IPTG). After the cell lysate is prepared, the fusion protein is purified in one step using the N-terminus 6xHis tag’s high affinity to the nickel chelating resin, Ni-NTA, and the efficient elution by imidazole. If desired, the affinity tag can be removed by using pQE30-Xa plasmid which contains the FXa proteolytic recognition site downstream from the His₆ tag.

The E. coli strains

Any E. coli host strain containing both the expression (pQE) and the repressor (pREP4) plasmids can be used for the production of recombinant proteins. This kit, the QIAexpress System, uses E. coli strains M15 and Sg13009; both harbor the pREP4 plasmid, which is maintained by including kanamycin in the growth media. Both strains permits high-level expression and are easily handled. Proteins that are poorly expressed in M15 may be produced in high levels in SG13009. Both strains are derived from E. coli K12. E. coli strains that harbor the lacI^q mutation, such as JM109 (Stratagene), Xl1 Blue and TG1, produce enough lac repressor to efficiently block transcription, and are ideal for storing and propagating pQE plasmids. These strains can also be used as expression hosts for expressing nontoxic proteins, but they may be less efficient than the M15 and expression is regulated less tightly than in the strains harboring the pREP4 plasmid.

The chelating resin Ni-NTA

Nitrilotriacetic acid (NTA) that was developed at Qiagen is a chelating adsorbent that occupies four of the six ligand-binding sites in the coordination sphere of the nickel ion, leaving two sites free to interact with the histidine residues. The NTA binds metal ions far more stably than any other chelating resin and retains the ions under a wide range of conditions, including stringent wash conditions. Therefore, the NTA matrices can bind His₆-tagged proteins very tightly, allowing even low level expressed proteins (<1%) to be purified to greater than than 95% homogeneity in a single step. The Ni-NTA is coupled to Sepharose® CL-6B and offers high binding capacity with minimal nonspecific binding. This agarose can be used for purification via batch, column or low-pressure liquid chromatography. The Ni-NTA agarose capacity is sufficient for the binding of 5-10 mg of His₆-tagged protein per 1 mL resin. It is recommended to purify only one type of protein on the same batch and after 5 purifications to use a fresh resin.

Additional kit contents

By purchasing this kit, you will have everything you need to purify your protein, be it soluble or insoluble. In addition to the above, you will also get the following reagents: pREP4 plasmid; control expression plasmid pQE40; 1M imidazole stock solution; 1 mL of 1M IPTG (powder); urea; guanidine hydrochloride and sodium phosphate stock solution. The kit is also supplied with 1 and 5 mL disposable columns. The QIAexpressionist handbook is comprehensive; it elaborates on every aspect and provides near perfect instructions to guide you rationally through the multiple steps to obtain high homogeneity of your purified protein. Protein expression begins with constructing expression clones, continues with the expression of His₆-tagged proteins, followed by the purification on the Ni-NTA resin. The handbook contains a long list of ordering information for products that you may need, including those for performing PCR and primers for sequencing of the pQE plasmid. If you aim to purify recombinant proteins expressed in an E. coli culture ranging from milliliters to several liters, you will find this kit very useful. It covers the most important considerations and troubleshooting of cloning, expression and purification. It would be good if in the section on expression, the handbook elaborated more on the varying conditions under which it is recommended to express proteins of different natures, sizes, functions, toxicities and compartmentalizations. Despite this, I recommend the kit for being a complete solution for purifying recombinant proteins to high homogeneity in a relatively short period of time for an affordable price.