Isolation of epitope-tagged proteins from bacterial cells normally involves the use of specialized equipment like a French Press or sonicator. Unfortunately, not all labs that are interested in expressing proteins using bacteria have access to equipment of this type, especially if the need is infrequent, part of a pilot project or for gathering preliminary data. To meet the needs of researchers in these situations and to make small pilot expression projects easy to complete quickly, Pierce has produced a kit which allows small purifications of GST-tagged proteins in a minimum amount of time without specialized equipment.
The B-PER GST Fusion Protein Purification Kit relies on Pierce’s proprietary B-PER lysis reagent to effectively lyse bacterial cells without specialized equipment. Other homemade lysis solutions are available, but due to their harsh nature, they have the potential to interfere with downstream applications such as affinity purifications. Thus, many labs rely on mechanical means to disrupt cells and release soluble proteins. The B-PER reagent is based on a much milder treatment which produces soluble protein in buffered solution which is compatible with affinity purification, in this case, using an immobilized glutathione column.
Expression of the recombinant protein is induced in up to a 250 ml culture as appropriate for the specific expression vector. Cells are harvested by centrifugation and then resuspended in the B-PER reagent. Following a room temperature incubation, the resulting slurry is loaded directly onto the provided glutathione-agarose columns. The unbound proteins are washed from the bound GST-tagged proteins in a series of washes. Then the GST-tagged protein is eluted from the column by incubation with free glutathione.
The purified protein is now ready for most downstream applications, for instance, it can be mixed directly with sample buffer and run on a polyacrylamide gel. One thing to note however, is that when you analyze the purified proteins using SDS-PAGE and coomassie staining is that there is seldom a single clean band. In our hands, there were often contaminating bands present that may interfere with subsequent downstream applications. In cases where highly purified protein is needed, a more rigorous purification may be required. However, if a small amount of contaminating bacterial protein will not interfere with other applications, for instance -- western blotting, the kit provides quite acceptable yields of the specific GST-tagged protein.
Overall, this kit provides an excellent option for directly purifying significant quantities of GST-tagged proteins from bacterial cultures quickly and easily. The yields are good, the purity acceptable and the kit quite easy to use. For anyone making a first foray into the world of bacterial protein expression, this kit provides an excellent starting point.
B-PER GST Fusion Protein Purification Kit From Thermo Scientific Pierce Protein Research Products
Straightforward kit that provides straightforward lysis of bacterial cells and isolation of GST-tagged recombinant proteins.
Yields are variable and the purity of the isolated protein may not be sufficient for downstream applications.
The Bottom Line
A quick, easy way to isolate GST-tagged proteins; good for small scale and those new to the technique.