Aptamers in protein purification
Protein expression and purification is a standard technique in most molecular biology labs and facilities, allowing detailed characterisation of protein structure and function.
To facilitate purification from an overexpression system, proteins of interest are often fused with specific peptide chains to allow binding to column matrices. Popular peptide tags include His, allowing immobilisation to Ni-NTA treated beads, and Flag / Myc tags which are recognised by antibodies.
However, not all proteins are amenable to tagging with a fusion peptide, especially if the sequence encoding the protein is unknown. Moreover, often tags can interfere with activity and require cleavage, adding additional time and cost to experiments.
Similar to Flag and Myc tags, affinity chromatography relies on immobilisation of antibodies to a column support to trap a target of interest. The use of antibodies allows proteins to be isolated in their native form e.g. all appropriate post-translational modifications and can allow proteins to be isolated from primary cells, rather than overexpression systems.
Since Affinity chromatography is antibody dependent, it requires antibodies to have a high affinity to the target in order to isolate enough material for examination. Moreover, antibodies need to be highly specific to prevent isolating irrelevant material which may also impair downstream assays.
Once the antibody has captured the protein target, elution can also be a problem for antibodies. In many cases, the antibody is also released from the column along with the target which can mask the target when analysed via SDS-PAGE, particularly if the target is a similar molecular weight to an antibody chain. The elution process also requires the use of harsh chemicals which can denature the protein and preventing subsequent analysis.
Aptamer-Mediated Affinity Chromatography
The use of aptamers in affinity chromatography aims to help solve many of these problems associated with protein purification. By building in specific elution conditions, the aptamer can be engineered to bind the target with high affinity and specificity, yet release when desired.
Typical workflow of a custom AptaBind project:
- Identify your target
- Raise aptamers recognising your target
- Build in an elution module, allowing release under specific conditions
- Immobilise the aptamer to column resin ready for target purification from any cell type
Please click on the link below to read more about AptaBind:
Aptamer Mediated Affinity Chromatography (AMAC)
Custom Developed Purification / Chromatography Columns
Aptamer modification also permits immobilization of the aptamer on to the surface of a compatible resin. The immobilised aptamers can then be used to capture specific targets from complex mixture by Aptamer Mediated Affinity Chromatography (AMAC). The high specificity of aptamers for their respective targets means that a higher degree of purification should be possible than can be achieved by traditional affinity chromatography processes. This also means that native proteins may be expressed and purified without the need for additional tags (e.g. Hexa-Histidine tags). This would also negate the need for cleavage and additional purification steps.
Unlike antibody based immuno-depletion columns, aptamer columns are more readily regenerated and may be reused several times without significant loss of function. In addition to this, specific elution steps may be built in to the selection process to facilitate these regeneration steps. Purification columns of this nature are now in use for preconcentraion / purification of various environmental / food contaminants before analysis by mass spectrometry.