Advantages of gentle ImmunoAffinity Chromatography with Softag™ mAbs:
- Elute under stabilizing conditions
- Retention of enzyme activity
- Can wash with salt or polyol alone to remove non-specifically bound proteins
- Ability to isolate multi-subunit complexes
- Ability to isolate enzyme-associated proteins
- Produce homogeneous enzyme suitable for crystallization
- Can transfer advantages through epitope tag to fusion protein
- Cost-effective elution
Possible uses for researcher
- Recombinant protein detection (Western)
- “Gentle” protein purification
- Protein complex isolation
- Immunoprecipitation
- Protein-protein interaction
- Chromatin IP
- High-throughput protein purification system
Protein purification has and will play a key role in the Proteomics initiative. Purification involves the separation and isolation of proteins for study. While there are a number of ways to separate proteins, immunoaffinity chromatography is one of the most powerful protein purification procedures available. This method relies on the high affinity antibody-antigen interaction that enables highly selective binding of the protein of interest. Bound antigenic proteins can be unbound or “eluted” off from the antibody and then studied to identify the biological function of the protein and its interaction with other proteins and partners. Immunoaffinity chromatography has proven extremely useful for both biochemical laboratory scale and some larger-scale protein purification.
MAbs have several advantages for use in immunoaffinity chromatography. Once a monoclonal antibody producing cell line is established, it can be used to make a potentially unlimited supply of antibody with reproducible properties. MAbs also bind to a single binding site and have homogeneous binding and elution properties enabling absolutely uniform purification for the protein of interest.
Another wrinkle in the immunoaffinity-chromatography-purification approach is the use of Epitope Tags. An epitope tag is small segment of a protein, the genetic sequence of which is known AND the segment (epitope) to which a known mAb binds. Ideally, these segments are 15 amino acids or less to facilitate cloning. The tags are initially attached to the end of the protein of interest and can be released by proteases or left in place. These tags enable the needed binding between the protein of interest and the mAb. Once the protein of interest and tag are eluted from the mAb, you have the purified protein.
Sounds wonderful, doesn’t it? Unfortunately, there are a few catches to this marvelous scenario. Because of the strength of the antigen-antibody interaction it is very difficult to elute the antigen or epitope tag from the bound mAb. It is not uncommon to employ quite harsh elution conditions, such as extreme pH values, denaturing agents, or chaotropic salts that disrupt protein structure. The elution conditions often damage proteins, resulting in very low yields (or none at all) of fully active, purified protein. In none of the protein purification variations is it possible to isolate any proteins normally associated with the protein-of-interest in its active, functioning state. These conditions also reduce the lifetime of the antibody column. In addition, the cost and complexity of mAb development has limited use of mAbs for protein purification. So while the epitope tag approach can be very useful and is extensively used by researchers and biotech companies, it is far from the ideal method of purifying proteins in its current guise.
And this is where NeoClone is positioned to provide a very elegant solution. NeoClone utilizes a special class of mAbs that are refered to as “polyol-responsive mAbs” (PR-mAbs). PR-mAbs have the ability to non-destructively release the protein-of-interest to which they are bound so the protein retains its full biological activity AND any other proteins with which it is functionally associated. There has also been developed a corresponding class of epitope tags (called Softag™), which bind to these antibodies. We have licensed these exclusively through the Wisconsin Alumni Research Foundation (WARF) and are now making them available to researchers. The possible uses of this remarkable class of mAbs and their technological advantages are highlighted below.