B-PER 6xHis Fusion Protein Purification Kit From Thermo Scientific Pierce Protein Research Products

Production of recombinant protein using bacterial expression systems is a powerful tool available to modern researchers. The ability to utilize E.coli to produce large quantities of a desired protein is dependent upon a technique for purifying the protein of interest from the contaminating bacterial proteins. For this application, recombinant proteins are tagged with a specific amino acid sequence which allows it to be purified through the use of binding substrates.

The Pierce B-PER 6xHis Fusion Protein Purification Kit relies on a proprietary lysis reagent to efficiently lyse bacterial cells without the use of expensive equipment like sonicators or a French Press. This is a definite advantage for labs new to bacterial expression and protein isolation as it comes complete with everything needed to extract total bacterial protein, and then purify the 6xHis tagged recombinant protein from the remaining bacterial proteins.

In most cases, purifications for small scale users, including labs new to bacterial protein expression, require modest amounts of protein. Pierce claims that this kit is capable of purifying greater than 10 mg of 6xHis tagged protein from a 250 ml bacterial culture. Yields in this range are more than sufficient for most applications at the research lab level. If larger quantities of protein are required, it is relatively easy to purify multiple cultures in a short period of time. In fact, a single purification requires approximately two hours and in theory, multiple purifications could be done simultaneously. The kit contains sufficient columns for doing five purifications; however, their protocol states that the columns can be washed and re-used. Thus, it may be possible to stretch the kit to perform ten or more purifications.

The histidine-tagged proteins are purified away from the contaminating bacterial proteins by column chromatography utilizing a nickel-chelated agarose matrix. The columns selectively bind the string of histidine residues in the recombinant protein while allowing the non-tagged proteins to flow throw. Pierce claims to be able to achieve high levels of purity directly from the column. In practical applications, we observed purity levels that were less than advertised. This was largely due to a set of commonly purified bands which appeared in purifications of multiple recombinant proteins. This contamination is most likely due to the presence of native proteins which contain multiple adjacent Histidine residues and thus, co-purify with the specific recombinant protein.

In our hands, the B-PER 6xHis Fusion Protein Purification Kit provided a simple-to-use tool for the isolation and purification of his-tagged recombinant proteins. Yields were lower than expected, typically in the 2-5 mg range per 250 ml culture. However, every expression construct can vary in its characteristics and with optimization of growth and expression profiles, it would probably be possible to increase these yields. Further, in all cases, purity was less than suggested based on the presence of the ubiquitous contaminating bands mentioned previously. Again, it may be possible to eliminate these contaminating proteins from the purification through the use of more stringent washes, or by optimizing the bacterial strains used for expression.

The bottom line for this kit is that it provides a valuable tool for rapidly and inexpensively purifying bacterial recombinant proteins. However, depending on the downstream application, this kit may not provide sufficient purity in and of itself and may require that further steps be performed to obtain sufficiently pure protein.