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  • Title: Selection of aptamers for a non-DNA binding protein in the context of cell lysate.
    Author: Kanoatov M, Javaherian S, Krylov SN.
    Journal: Anal Chim Acta; 2010 Nov 29; 681(1-2):92-7. PubMed ID: 21035608.
    Abstract:
    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.
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