Fusion proteins conjugated to glutathione-S-transferase (GST) are commonly used as antigens for antibody production or for GST pull-down assays. When a recombinant protein is expressed in bacteria, the initial cell lysate contains high levels of the induced protein along with several additional proteins. The desired protein is purified from the lysate by binding and eluting it from a glutathione resin. Before purification, the cells must be lysed in order to extract the protein. There are mechanical methods of lysing bacteria, but not all labs have access to the necessary equipment. Moreover, a sonicator generates heat that can degrade the protein. As an alternative, Novagen offers a chemical extraction reagent that requires only a shaker as part of their purification kit.
Recently, I needed to express and purify GST-fusion proteins for antibody production. Since neither I nor anyone else in the lab had done any protein expression work before, I wanted a kit that would guide me through the procedure and simplify the purchasing of reagents. After expressing a GST-tagged protein in bacteria, the BugBuster GST-Bind kit supplies all of the necessary reagents for extracting and purifying the protein. It includes the glutathione resin, washing buffer, reduced glutathione for elution, and the detergent-based extraction reagent. The only reagents that are not included are the optional lysozyme and protease inhibitors.
Without a sonicator or French press in the lab, the detergent-based BugBuster reagent was far more convenient than bringing my samples to a colleague’s lab. When used with Benzonase nuclease, the extract loses some of its viscosity and can easily flow through the affinity column. The benefit of the nuclease is that a fast flow rate lessens the chance of degradation, since the eluate can be properly stored faster. The extraction reagents are also compatible with protease inhibitors if desired. Another advantage is that the GST resin is supplied as a 50% slurry that does not need to be swollen. Columns are supplied but are not prepacked, allowing researchers the flexibility to perform a batch purification or column chromatography using an appropriate volume of resin. In addition, the column can be reused a few times without regeneration if properly stored. The company recommends performing the purification at room temperature, which makes the process more convenient. I found that my results were similar regardless of whether I kept my samples at room temperature or 4ºC during the purification. At the end of the procedure, I dialyzed the protein into a suitable buffer and then my antigen was ready to send away for antibody production.
While I have no complaints about the product itself, it is important to remember some proteins are very difficult to purify on a GST resin. I had one fusion protein that flowed through the resin, likely due to an improperly folded GST tag. Another protein was nearly impossible to elute from the resin. A third worked beautifully, providing a high yield of purified protein. Since it is not always possible to predict how a protein will behave, there is a risk that this approach may not be suited to a protein of interest.
The kit is probably an appropriate choice for researchers who are purifying a recombinant protein for the first time and do not have any of the necessary reagents. The reagents are also sold individually for researchers who wish to purchase the BugBuster reagent, Benzonase nuclease, or resin individually. This option would be more appropriate for researchers who prefer mechanical lysis of the cells or those who already have GST beads. Since Novagen specializes in expression and purification of recombinant proteins, they have most of the reagents necessary to optimize the yield of soluble recombinant protein and to aid in purifying difficult proteins.
Tara Lauriat
Graduate Student
Mount Sinai School of Medicine
New York, NY