Using Aptamers for Protein Scale-up

Abstract

Purifying and concentrating proteins is fundamental to biomedical research for both diagnostics and therapeutics purposes. Several methods can be used for protein purification, ranging from simple chemical methods to more advanced chromatography techniques. We looked at improving research solutions for analysing and concentrating proteins using DNA aptamers for specific soluble proteins. Aptamers can be combined with conventional methods like affinity chromatography by attaching an aptamer to silica or magnetics beads. The protein is bound and subsequently eluted from the bead to yield purified protein. We have developed computational approaches for developing aptamers against any protein which eliminates the need for making a recombinant protein with a tag, allowing for the purification of more native proteins. In this study a computational approach was used to determine the binding sites between an existing reference aptamer (R_apt) to a well characterised protein, Bovine Serum Albumin (BSA). We found that R_apt binds to BSA specifically at domains I and III. Following this we characterised the binding of R_apt to BSA in vitro using the intercalating dye, SYBR Green I, to show a dissociation constant (KD) 0.02 {micro}M. The R_apt was modified by adding 5 adenosines to the 5 end to make a polyA tail. This aptamer with a modified polyA tail, named Ni_apt, allows binding to Ni-NTA magnetic beads. The Ni_apt had a dissociation constant (KD) to R_apt to be 0.12 {micro}M. Lastly, we utilised Ni-NTA magnetic beads coupled with the Ni_apt aptamer to bind and purify BSA from a concentrated solution. We recovered 20.7% of the BSA using our protocol. In future developments, we aim to extend our technology based on this foundation to target proteins with therapeutic or diagnostic potential, such as extracting and concentrating immunoglobulins, antibodies and high value proteins.