Preparation of immunoaffinity column for rapid purification of human DNA polymerase delta and its subassemblies
Mammalian deoxyribonucleic acid (DNA) polymerase delta (pol δ) is well characterized as a tightly associated heterotetrameric complex. It is thought to play a central role in chromosomal DNA replication and various DNA repair processes. However, the availability of highly purified active pol δ becomes one of the major barriers for its in-depth functional and structural analysis. In this work, a powerful immunoaffinity column was prepared. The human DNA pol δ and its subassemblies were reconstituted with a novel MultiBac system, over-expressed in insect cells, and followed by purification using immunoaffinity chromatography in combination with ion-exchange chromatography. Starting from 500 ml of infected Sf-9 cells, as much as 5 mg of recombinant pol δ with different subunit combinations was isolated near homogeneity with active forms that were conformed by the assays both on sparsely primed poly (dA)/oligo (dT) template-primer and on singly primed M13 DNA template. Thus, our home-made immunoaffinity column provides a significant advance in the isolation of pol δ, allowing its facile isolation from over-expressed insect cells or natural source in good yield and high purity.
Keywords: DNA polymerase delta, immunoaffinity chromatography, reconstitution, MultiBac system, subassemblies