Production of recombinant human DNA polymerase delta in a Bombyx mori bioreactor

Abstract

Eukaryotic DNA polymerase δ (pol δ) plays a crucial role in chromosomal DNA replication and various DNA repair processes. It is thought to consist of p125, p66 (p68), p50 and p12 subunits. However, rigorous isolation of mammalian pol δ from natural sources has usually yielded two-subunit preparations containing only p125 and p50 polypeptides. While recombinant pol δ isolated from infected insect cells have some problems of consistency in the quality of the preparations, and the yields are much lower. To address these deficiencies, we have constructed recombinant BmNPV baculoviruses using MultiBac system. This method makes the generation of recombinant forms of pol δ containing mutations in any one of the subunits or combinations thereof extremely facile. From about 350 infected larvae, we obtained as much as 4 mg of pol δ four-subunit complex. Highly purified enzyme behaved like the one of native form by rigorous characterization and comparison of its activities on poly(dA)/oligo(dT) template-primer and singly primed M13 DNA, and its homogeneity on FPLC gel filtration. In vitro base excision repair (BER) assays showed that pol δ plays a significant role in uracil-intiated BER and is more likely to mediate LP BER, while the trimer lacking p12 is more likely to mediate SN BER. It seems likely that loss of p12 modulates the rate of SN BER and LP BER during the repair process. Thus, this work provides a simple, fast, reliable and economic way for the large-scale production of human DNA polymerase δ with a high activity and purity, setting up a new platform for our further research on the biochemical properties of pol δ, its regulation and the integration of its functions, and how alterations in pol δ function could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability.

Figure 1. A strategy for assembling multigene expression cassettes in pFBDM.

Four genes, p125, p50, p68, and p12, were inserted into MCS1 between BamHI and XbaI sites, respectively. The expression cassette containing p125 was excised by digestion with AvrII and PmeI (boxed) and placed into a multiplication module (M) of a construct containing p50 via BstZ17I/SpeI sites. Followed the same logic, a pFBDM-[p125-p50-p68] was generated by an insertion of the expression cassette containing p125 and p50 into a construct containing p68. Finally, a recombinant transfer vector pFBDM-[p125-p50-p68-p12] was generated in which each subunit is under the control of individual polyhedrin promoter.

Figure 2. A schematic outline for generation of recombinant virus and its expression in silkworm larvae.

The obtained recombinant transfer vector pFBDM-[p125-p50-p68-p12] was introduced into BmNPV bacmid DNA in BmDH10Bac E. coli cells. Colonies containing bacmid carrying integrated four-gene expression cassettes were identified by blue/white screening. Bacmid DNAs were isolated from selected white phenotypes. The recombinant BmNPV viruses were generated by the transfection of BmN insect cells with isolated bacmid DNAs. The four subunit pol δ complex was then expressed and assembled in silkworm larvae by the infection of larvae with prepared recombinant viruses.

Figure 3. Western blotting analysis for the generation of the recombinant BmNPV viruses in BmN cells.

Eight bacmid DNAs, isolated from the white clones and verified by PCR analysis, were transfected into BmN cells. After 5 days post-transfection, each supernatant was harvested as the viral stocks and the cell pellets were analyzed by Western blotting using the antibodies specific for the individual subunit as described under “Materials and Methods”. M indicates the positions in kDa of protein markers. The lanes from 1 to 8 show eight independent transfections of BmN cells with recombinant BmNPV bacmid DNAs. Pol δ four subunits are marked by arrows.

Figure 4. Purification of recombinant human pol δ heterotetramer by immunoaffinity chromatography.

Infected larval hemolymph was centrifuged and divided into supernatant and pellet fractions. The supernatant fraction was subjected to immunoaffinity column. The lysates (BC), flow-through (FT), wash (W), and eluted fractions were assayed for DNA polymerase activity in the presence (square) and absence (diamond) of PCNA (panel A), and analyzed by 12.5% SDS-PAGE followed by Western blotting (panel B) using the antibodies specific for the individual subunit as described under “Materials and Methods”. A parallel purification for the pellet fraction was also performed. The activities were assayed (panel C) and Western blotting analysis was carried out (panel D).

Figure 5. Purification of recombinant pol δ heterotetramer by Mono Q chromatography from supernatant fraction of hemolymph.

The peak fractions (fractions 6–21) from the immunoaffinity chromatography step in which the column was applied with supernatant fraction of hemolymph were combined and passed through a Mono Q column. The lysates (BC), flow-through (FT), and eluted fractions were analyzed by 12.5% SDS-PAGE followed by Western blotting (panel A) or Coomassie Blue staining (panel B).

Figure 6. Characterization of recombinant pol δ heterotetramer complex.

Panel A: Coomassie Blue protein stain of the pol δ enzymes used in this experiment. Lane 1 shows the recombinant pol δ enzyme isolated from supernatant fraction of infected silkworm larvae hemolymph after Mono Q chromatography step. Lane 2 shows the native pol δ enzyme isolated from cultured Hela cells by immunoaffinity chromatography. Panel B: A direct side-by-side comparison of recombinant pol δ enzyme with the most highly purified native pol δ enzyme from Hela cells on its specific activities and response to PCNA (▪, native pol δ with PCNA; ▴, recombinant pol δ with PCNA; ⋄, native pol δ without PCNA; ×, recombinant pol δ without PCNA). Panel C: Analysis of DNA products synthesized by recombinant pol δ holoenzyme on primed M13 DNA as described under “Materials and Methods”. Horizontal bars (Lane M) on the left indicate the positions in kb of DNA markers. Lane 1–2 show 200 fmoles of native pol δ isolated from Hela cells as the positive controls in the presence (Lane 1) and absence (Lane 2) of PCNA. The Lanes from Lane 3 to 7 show increasing amounts of recombinant pol δ enzyme (fmole) containing 0, 50, 100, 150, and 200, respectively. Panel D: Equal amounts of native form pol δ enzyme from Hela cell preparation analyzed in holoenzyme assay. The lanes from left to right show the increasing amounts of native enzyme of 50, 100, 150, 200 fmoles.

Figure 7. Molecular weight determination of recombinant pol δ heterotetramer complex.

A 250 µl of sample from Mono Q peek fraction 15 was passed through a Superdex 200 column precalibrated with molecular weight standards. Panel A: The fractions across the peak of polymerase activity were run on 12.5% SDS-PAGE and Western-blotted with the indicated antibodies. The pol δ four subunits are marked by arrows. Panel B: The diagram shows the calibration of the column that was used to estimate the molecular weight. The standards used here were: a, thyroglobulin (667,000); b, ferritin (445,000); c, catalase (232,000); d, aldolase (158,000); e, bovine serum albumin (67,000). The elution position of the recombinant heterotetramer complex is indicated by a vertical arrow. A calibration curve was plotted by the logarithm of molecular weights versus calculated Kav.

Figure 8. Contribution of pol δ to SN and LP BER of uracil-containing plasmid DNA.

Panel A: A schematic representation of the uracil-containing plasmid DNA with KpnI and XhoI restriction-enzyme sites (underlined). After BER reaction, the repaired products were restricted with indicated enzymes, and digested DNA fragments were separated on 16% denaturing polyacrylamide gel containing 8 M urea. DNA fragment sizes and descriptions of DNA synthesis products are indicated. Panel B and C: Autoradiograph of gel electrophoresis to measure in vitro BER products. The BER reaction was performed with cellular extracts (B), from wild-type MEF (lane 1, 2), pol β −/− (lane 3, 4) cells, pol δ–depleted wild-type MEF (lane 5, 6), and pol δ–depleted pol β −/− (lane 7, 8). The BER reaction was performed with purified enzymes (C), pol δ4 (lane 1, 2) and pol δ3-p12 (lane 3, 4). Lane M indicates a ³²P labeled 25-bp oligonucleotide as a DNA maker. Panel D: The ratio of SN BER and LP BER with purified pol δ4 (upper panel) and pol δ3-p12 (lower panel). The amount of the SN BER product was calculated by subtracting the radioactivity of the 16-bp fragment from that of the 25-bp fragment.