Purification and Characterization of Human DNA Ligase IIIα Complexes After Expression in Insect Cells

Abstract

With improvements in biophysical approaches, there is growing interest in characterizing large, flexible multi-protein complexes. The use of recombinant baculoviruses to express heterologous genes in cultured insect cells has advantages for the expression of human protein complexes because of the ease of co-expressing multiple proteins in insect cells and the presence of a conserved post-translational machinery that introduces many of the same modifications found in human cells. Here we describe the preparation of recombinant baculoviruses expressing DNA ligase IIIα, XRCC1, and TDP1, their subsequent co-expression in cultured insect cells, the purification of complexes containing DNA ligase IIIα from insect cell lysates, and their characterization by multi-angle light scattering linked to size exclusion chromatography and negative stain electron microscopy.

Keywords: Affinity chromatography; Bacmid; Baculovirus; Insect cells; Ion exchange chromatography; Multiple angle light scattering; Negative stain electron microscopy; Size exclusion chromatography.