Nurit Becker, Ph.D.- Human Protein Biology Department Head/R&D, Sigma-Aldrich International GmbH, Rehovot, Israël, An affiliate of Merck KGaA, Darmstadt, Germany; Daniel Taglicht, Ph.D.- Department Head | Protein Expression & Purification/R&D, Sigma-Aldrich International GmbH, Jerusalem, Israël, An affiliate of Merck KGaA, Darmstadt, Germany
When proteins are overexpressed in cells for studying these proteins either within the cellular host or as a purified preparation, it is quite common that researchers add one or more of a variety of short peptide tags or larger domains, to assist in the planned experiment. In general, the short peptide tags are added to assist in detection, capture and purification of the protein of interest, while the larger protein domains are used to enhance protein solubility. The most common peptide tags are 6xHis, FLAG®, HA, cMyc, V5. The Most commonly used protein domains are GST (Glutathione S-transferase), MBP (Maltose Binding protein), and SUMO (Small Ubiquitin-like Modifier).
Beads and resins to capture and purify proteins
The things to consider when developing a reliable and robust protein purification method include choosing the right column for chromatography, calibrating optimal binding and elution conditions and establishing the sequence of purification steps. The method must enable the intended use of the protein. For analytical purposes, a main consideration is whether the protein must remain functional for a subsequent enzymatic assay, or be merely detectable by, for example, immunoblotting or MS analysis. Sample-sized amounts can be thoroughly purified with affinity-tagged magnetic beads in a short, single-step workflow that achieves high yields. ExtrAvidin®, Anti-HA, Anti-C-MYC or Anti-V5 are examples of such beads.
High-quality preparative purification of substantial amounts of a protein are a different challenge than small-scale sample preparation. The protein’s activity must usually be maintained. The most common approach involves the 6-histidine-tag, which allows purification on Ni affinity resins, and the GST domain for purification on glutathione affinity resins. Both require only relatively mild elution conditions, using imidazole and glutathione, respectively. The harsher elution methods of antibody-based capture reagents are mostly unsuitable for active-protein purification. An exception to this is when a lower affinity antibody is available that allows the tagged protein to elute by adding a short synthetic peptide that competes out the captured protein. A good example of this are FLAG® reagents based on the anti-FLAG® M2 monoclonal antibody. To elute a protein that binds to an antibody tightly, a gentle method is to insert between the affinity tag and the protein of interest a short cleavage site that is recognized by a site-specific enzyme such as the TEV, HRV 3C, SUMO or thrombin proteases.
Elution methods using epitope peptides and cleavage proteases
Epitope peptides serve as elution reagents by simply competing with the protein to bind the beads. This method allows elution under favorable buffer conditions, requiring no pH change or further chemicals to be added, although a peptide elimination step might subsequently become necessary. Elution using chemicals, imidazole or glutathione for example, is relatively straightforward and cost-effective. It is easy to align to a robust process and is as effective as any affinity chromatography process. On the other hand, chemical elution requires a specific tag or fusion protein to be expressed with the target protein and an added downstream process to remove the chemical. Moreover, many proteins can degrade if exposed to some of the typically used chemicals for long periods of time.
Oftentimes the affinity tag or carrier protein needs to be removed as it may interfere with the activity of the purified protein. In fact, various industries must adhere to strict regulatory guidelines that require tag-free proteins or tag removal for downstream purification. To facilitate the proteolytic separation of the tag and the protein of interest, one of several protease recognition sequences is often introduced between the affinity tag or carrier domain and the protein of interest. Importantly, these tag-removal proteases must be specific and have a recognition sequence that does not appear in the protein itself. Commonly used cleavage proteases include TEV, HRV 3C, thrombin and SUMO. SUMO protease differs in that it does not recognize a short recognition sequence but rather the SUMO domain’s secondary structure. The enzyme removes the complete SUMO domain without leaving any remaining amino acids on the protein. Therefore, SUMO protease is usually the best solution when an authentic N-terminal sequence is required.
See our extensive range of products and solutions for protein purification in biological and biomedical research SigmaAldrich.com/ProteinPurification
See our extensive range of products for protein and antibody purification, which includes magnetic beads, agarose gels, enzymes, peptides, antibodies, vectors, coated plates, FLAG® purification tools and more. SigmaAldrich.com/ProteinChromato
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