Proliferating cell nuclear antigen-agarose column: A tag-free and tag-dependent tool for protein purification affinity chromatography

Abstract

Protein purification by affinity chromatography relies primarily on the interaction of a fused-tag to the protein of interest. Here, we describe a tag-free affinity method that employs functional selection interactions to a broad range of proteins. To achieve this, we coupled human DNA-clamp proliferating cell nuclear antigen (PCNA) that interacts with over one hundred proteins to an agarose resin. We demonstrate the versatility of our PCNA-Agarose column at various chromatographic steps by purifying PCNA-binding proteins that are involved in DNA Replication (DNA polymerase δ, flap endonuclease 1 and DNA ligase 1), translesion DNA synthesis (DNA polymerases eta, kappa and iota) and genome stability (p15). We also show the competence of the PCNA-Agarose column to purify non-PCNA binding proteins by fusing the PCNA-binding motif of human p21 as an affinity tag. Finally, we establish that our PCNA-Agarose column is a suitable analytical method for characterizing the binding strength of PCNA-binding proteins. The conservation and homology of PCNA-like clamps will allow for the immediate extension of our method to other species.