Proliferating cell nuclear antigen-agarose column: A tag-free and tag-dependent tool for protein purification affinity chromatography
KAUST DepartmentBiological and Environmental Sciences and Engineering (BESE) Division
KAUST Grant NumberCRG3
Permanent link to this recordhttp://hdl.handle.net/10754/656035
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AbstractProtein 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.
CitationTehseen, M., Raducanu, V.-S., Rashid, F., Shirbini, A., Takahashi, M., & Hamdan, S. M. (2019). Proliferating cell nuclear antigen-agarose column: A tag-free and tag-dependent tool for protein purification affinity chromatography. Journal of Chromatography A, 1602, 341–349. doi:10.1016/j.chroma.2019.06.008
SponsorsThis work was supported by King Abdullah University of Science and Technology through core funding and Competitive Research Award [CRG3 to S.M.H.].
JournalJournal of Chromatography A