Aptamer facilitated purification of functional proteins.

DNA aptamers are attractive capture probes for affinity chromatography since, in contrast to antibodies, they can be chemically synthesized and, in contrast to tag-specific capture probes (such as Nickel-NTA or Glutathione), they can be used for purification of proteins free of genetic modifications (such as His or GST tags). Despite these attractive features of aptamers as capture probes, there are only a few reports on aptamer-based protein purification and none of them includes a test of the purified protein's activity, thus, leaving discouraging doubts about method's ability to purify proteins in their active state. The goal of this work was to prove that aptamers could facilitate isolation of active proteins. We refined a complete aptamer-based affinity purification procedure, which takes 4 h to complete. We further applied this procedure to purify two recombinant proteins, MutS and AlkB, from bacterial cell culture: 0.21 mg of 85%-pure AlkB from 4 mL of culture and 0.24 mg of 82%-pure MutS from 0.5 mL of culture. Finally, we proved protein activity by two capillary electrophoresis based assays: an enzymatic assay for AlkB and a DNA-binding assay for MutS. We suggest that in combination with aptamer selection for non-purified protein targets in crude cell lysate, aptamer-based purification provides a means of fast isolation of tag-free recombinant proteins in their native state without the use of antibodies.

Beloborodov SS ，Bao J ，Krylova SM ，Shala-Lawrence A ，Johnson PE ，Krylov SN ... -  《-》

Selection of aptamers for a protein target in cell lysate and their application to protein purification.

Javaherian S ，Musheev MU ，Kanoatov M ，Berezovski MV ，Krylov SN ... -  《-》

Selection of aptamers for a non-DNA binding protein in the context of cell lysate.

Aptamer-facilitated Protein Isolation from Cells (AptaPIC) is a recently introduced method that allows, in particular, generation of aptamers for a protein target in a context of a crude cell lysate. The approach enables efficient, tag-free, affinity purification of target proteins which are not available in a pure form a priori, and for which no affinity ligands are available. In the proof-of-principle work, AptaPIC was used to develop aptamers for and purify MutS, a DNA mismatch repair protein. The DNA-binding nature of MutS raised concerns that AptaPIC was not a generic technique and could be inapplicable to protein targets that do not possess native nucleic acid-binding properties. Here we prove that these concerns are invalid. We used AptaPIC to generate pools of aptamers for human Platelet-Derived Growth Factor chain B (PDGF-B) protein, a non-DNA binding protein, in the context of a bacterial cell lysate, and subsequently purify it from the same lysate. Within a small number of rounds, the efficiencies of aptamer selection were similar in conventional Systematic Evolution of Ligands by Exponential Enrichment (SELEX) for pure protein and in AptaPIC for protein in the cell lysate. The conventional selection approach resulted in an aptamer pool with an EC(50) value of 2.0±0.1 μM, while the AptaPIC selection approach resulted in a pool with an EC(50) value of 3.9±0.4 μM. Our results clearly demonstrate that selection of aptamers for proteins in the cell lysate is not only realistic but also efficient.

Kanoatov M ，Javaherian S ，Krylov SN 《-》

Imidazole-free purification of His3-tagged recombinant proteins using ssDNA aptamer-based affinity chromatography.

Immobilized metal ion affinity chromatography (IMAC) is widely used for the purification of many different His6-tagged recombinant proteins. On the one hand, it is a powerful technique but on the other hand it has its disadvantages. In this report, we present the development of a unique ssDNA aptamer for the purification of His3-tagged recombinant proteins. Our study shows that stability of the His3-tag/H3T aptamer complex can be controlled by the sodium ion concentration. Based on this feature, we demonstrate that H3T aptamer resin was successfully employed for the purification of three out of four tested His3-tagged recombinant proteins from an E. coli total protein extract using imidazole-free buffers. Finally, we show that the purity of His3-tagged proteins is superior when purified with the help of the H3T aptamer in comparison with Ni-NTA resin.

Bartnicki F ，Kowalska E ，Pels K ，Strzalka W ... -  《-》

Kinetic capillary electrophoresis-based affinity screening of aptamer clones.

Yunusov D ，So M ，Shayan S ，Okhonin V ，Musheev MU ，Berezovski MV ，Krylov SN ... -  《-》