Thus, a protein complex containing four subunits was obtained as expected molecular masses. From 350 larvae, we yielded about 4 mg of four-subunit protein complex from the pooled Mono Q peak fractions 14�C17 purified to near homogeneity by our highly standardized protocol. A WY 14643 PPAR inhibitor summary for the specific activities and yield of preparations for purification of recombinant pol d holoenzyme was shown in table 1. Isolation of multi-subunit pol d from mammalian tissues has been extremely difficulties. One of them is the case of the p68 subunit which is prone to proteolytic nicking . Rigorous isolation of natural mammalian pol d resulted in no detectable p66 subunit in protein preparations . Even in the protocol which demonstrated co-purification of p66 with p125 and p50, the p66 subunit was subject to proteolytic degradation and stoichiometrically under-represented in the purified preparation . In this study, the four-subunit human pol d complex was reconstituted by overexpression in silkworm larvae with the recombinant BmNPV containing four subunit gene expression cassettes. Rigorous isolation of pol d activity from infected larvae led to the isolation of a near homogeneous pol d heterotetramer in an MK-2206 2HCl intact form. No degradation of the p68 subunit was observed in our preparation . Recent studies discovered a novel cellular response to DNA damage. Exposure of mammalian cells to UV light or alkylating agents led to the rapid degradation of the p12 subunit. Pol d was consequently converted from a heterotetramer in vivo to a trimer lacking the p12 subunit . The loss of the p12 did not result in the dissociation of the remaining subunits. This converted trimer in vivo could be isolated as an intact complex of p125, p50, and p68 using immunoaffinity chromatography with altered properties and behaviors when it encountered DNA base lesions . Previous studies also indicated that highly purified pol d enzymes by immunoaffinity chromatography were polydisperse on gel filtration. Further examination of the size of pol d by native gel electrophoresis gave results which indicated the existence of discrete complexes . There are major questions raised as to whether there exist subassemblies in vivo due to its intrinsic property of pol d. In this work, when the sample was applied with the pellet fraction of hemolymph containing cell debris and tissues, the pol d subassemblies were separated and appeared in Mono Q fractions .