How to develop aptamers for affinity chromatography
Reversible binding is essential for affinity chromatography. We discuss how to ensure you meet this critical property when developing aptamers for aptamer-based affinity chromatography applications.
In developing affinity chromatography ligands or wearable biosensors, you need more than high specificity from your aptamer. While antibody and aptamer developers talk about their ability to generate high target affinity and specificity, these properties aren’t necessarily focused on the end application and might not result in your desired performance.
The separation procedure of aptamer-based affinity chromatography
For affinity chromatography, one of your aptamer’s most essential properties is reversible binding. The formation of specific and reversible complexes of biological macromolecules is the basis of their separation, purification, and analysis by affinity chromatography.
In affinity chromatography, the affinity medium is equilibrated with the binding buffer to apply the sample in the optimum conditions for binding to the aptamer. The target molecule binds specifically, but reversibly, to the aptamer-resin and unbound material is washed through.
To recover the target protein, column conditions are changed to favor elution of the bound molecules. Elution is typically performed in a non-competitive format by changing the pH, salt concentration, divalent ion availability etc. The target molecule is then collected in a purified, concentrated form. Finally, the aptamer-resin can be cleaned and re-equilibrated with binding buffer.
Tailored discovery delivering functional aptamers for affinity chromatography
At Aptamer Group, we build these changes in target affinity into the discovery and development process to ensure the ligands you receive are effective at reversibly binding your target under the conditions you need. This includes ensuring target binding with a slow off-rate for aptamer-based affinity chromatography can be achieved in your feedstock for the loading phase, with elution under conditions to suit your target protein.
We have experience working with buffer changes as small as half a pH unit between binding and release or tailoring salt concentration according to your target to change ionic strength for target elution in aptamer-based affinity chromatography. These small changes can enable increased functional yields of fragile proteins. Equally, we can use divalent metal ion sensitivity, with requirements for magnesium for target binding in the loading phases, and using, for example, EDTA in the elution phase to chelate the divalent ions and prevent binding to the aptamer-resin.
All of these conditions can be varied according to your process and target requirements and are built into our discovery process, including performing selection for target binding in feedstock or counter-selection with tiny changes to ensure elution. Tailoring discovery according to your affinity chromatography process requirements ensures that your aptamer-based affinity chromatography process functions as you need and cuts down on the post-discovery development work required, saving you valuable time.
Aptamer-based affinity chromatography of a multi-subunit protein
Optimer binders were developed against a multi-subunit protein. Our discovery process was tailored to ensure binding in the partner’s feedstock, which was subsequently reversed by a gentle conditions to protect the protein complex. These binders were immobilized on a Sepharose resin for purification of the multi-subunit protein to support vaccine manufacture.
(a) Target protein purification was demonstrated in small-scale using Optimer immobilised on a Sepharose resin and an AKTA Explorer FPLC. (b) SDS-PAGE shows purification of the expected protein and retention of each of the protein subunits with no trace of contaminants