19th September 2019
Purification of functional proteins is a key procedure in development and production of biologics and often relies on traditional ion exchange, hydrophobic interaction or size exclusion chromatography. Although these techniques are relatively gentle, obtaining the desired level of purity requires many sequential and sometimes repeated purification steps. Affinity separation is also frequently used method because it offers a high yield and purity due to very selective interaction of the target protein. However, the process typically requires fusion of the target protein with an affinity tag such as His or GST, which may alter protein conformation and thus change its physiochemical properties.
Antibodies are commonly used as capture probes in tag-less purification method, however they come with their own limitations such as lengthy development process, sensitive to temperature and also undergo denaturation over the time. Aptamers offer the best alternative to antibodies for a tag-free purification method as they are more stable and can be easily designed using our automated selection process under costumer-defined conditions to yield highly specific aptamers (Figure 1).
Figure 1. Schematic representation of our in vitro selection process. The process can be tailored to include customer defined ‘loading’, ‘wash’ and ‘elution’ buffers.
At Aptamer Group, we have developed a process that benefits the purification of untaggedproteins in their active state using ‘aptamer mediated affinity chromatography’ (AMAC, Figure 2). The specificity of aptamers for their targets ensures that high levels of purity can be achieved from complex medium in a single AMAC step. Additionally the aptamers can be easily made with amine or thiol reactive groups to allow easy conjugation to the purification surface.
Protein loading, washing and elution steps are then carried out under customer-specified conditions. Mild elution conditions guarantee protein integrity following purification. In addition to these benefits, AMAC does not rely on affinity / solubility tags and therefore offers a simpler workflow for purifying ‘native’ proteins. Moreover, eluting under pre-determined conditions removes the need for harsh, denaturing reagents often required with other affinity ligands such as antibodies.
Figure 2. Schematic representation of protein purification using AMAC. Aptamer specific for the target is immobilized onto the resin (blue) so that the target molecule (red) is captured when the sample mixture is passed over the column. Wash steps are included to remove other sample components before eluting the target under customer-defined condition.
Overall, AMAC offers single-step purification using our highly specific aptamers, which increases the final yield of protein and dramatically reduces the purification time. This is considered important in industrial purification applications as production costs can be significantly reduced. For more information regarding the benefits of aptamers in protein purification, please contact us using the form below.