Aptamers help to facilitate specific protein purification
Purification of functional proteins is a key procedure in the development and production of biologics and relies on traditional ion exchange (IEX), hydrophobic interaction (HIC) or size exclusion chromatography (SEC). Although these techniques are relatively gentle, obtaining the desired level of purity requires many sequential and repeated purification steps. Affinity separation is also a frequently used method because it offers a high product and purity due to very selective interaction of the target protein.
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
- Target effectiveness can be hampered by needing an affinity tag e.g. HIS or GST
- Undergo denaturation over the time
Aptamers offers the best alternative to antibodies for tag-less purification method as they are:
- More stable
- Easily modified with a linker such as an amine or thiol group or biotin
- Moreover, due to their small size, aptamers can be more densely bound to a support, thereby increasing binding capacity
Taking advantage of highly specific aptamers, Beloborodov et al. developed a quick process that benefits the purification of tag-less proteins in their active state using biotinylated aptamer. The process follows as such:
- Multiple copies of protein-specific ssDNA aptamer were attached to magnetic beads via streptavidin-biotin bridge
- The aptamers served as capture probes for the target protein in a crude cell lysate
- The beads with captured target protein were then separated from the lysate by retaining the beads with a magnet and washing out the lysate
- Finally, the target protein was eluted with phosphate-buffered saline (PBS) supplemented with an additional electrolyte (NaCl or MgCl2) to increase the ionic strength.
Using this method, the authors successfully purified DNA repair proteins namely, AlkB and MutS from a crude bacterial cell lysate mixture. The purified eluates analyzed by SDS-MW established that the purity was more than 85%. Moreover, an enzymatic assay for AlkB and DNA binding assay for MutS showed that both proteins retained their specific activity.
Overall, aptamer-based purification method provides a means of fast isolation of tag-free recombinant proteins in their native state without the use of antibodies. Aptamers selective to specific target protein are readily modified to allow subsequent immobilization on a suitable resin. Protein loading, washing and elution steps are then carried out under customer-specified conditions.
At Aptamer Group, we have developed similar robust processes that benefit the purification of tag-less proteins in their active state using ‘aptamer mediated affinity chromatography’. For more information regarding the benefits of aptamers in protein purification, please get in touch.
Beloborodov, S.S., Bao, J., Krylova, S.A., Shala-Lawrence, A., John-son, P.E., & Krylov, S.N. (2018). Aptamer facilitated purification of functional proteins. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 1073, 201-206.