Overexpression of human DNA topoisomerase II alpha by fusion to enhanced green fluorescent protein.
DNA topoisomerase (topo) II alpha is a major target for many anticancer agents. However, progress towards understanding how these agents interact with this enzyme in human cells and how resistance to these agents arises is greatly impeded by difficulties in expressing this gene. Here, we report on achieving a high level of expression of a full-length human topo II alpha gene in human cells. We started with the topo II alpha cDNA driven by a strong cytomegalovirus promoter and transiently transfected HeLa cells. Although topo II alpha mRNA was consistently detected in transfected cells, no exogenous topo II alpha protein was detected. By contrast, when the same cDNA was fused to an enhanced green fluorescent protein (EGFP), we detected a high level of expression at both mRNA and protein levels. The exogenous topo II alpha was localized to cell nuclei as expected, indicating that the fusion protein is properly folded. Furthermore, overexpression of the EGFP-topo II alpha fusion protein increased the sensitivity of the transfected cells to teniposide, suggesting that it functions as the endogenous counterpart. Thus, in addition to being used as a gene tag, the GFP fusion approach may be generally applicable for expressing genes, such as topo II alpha, that are difficult to express by conventional methods.
Mo
Y Y
YY
University of Illinois at Chicago, USA.
Ameiss
K A
KA
Beck
W T
WT
D001483:Base Sequence; D002467:Cell Nucleus / Q000737:chemistry; D004250:DNA Topoisomerases, Type II / Q000032:analysis / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007202:Indicators and Reagents / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics*; D013713:Teniposide / Q000494:pharmacology; D059005:Topoisomerase II Inhibitors; D014162:Transfection
D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D059005:Topoisomerase II Inhibitors; D049452:Green Fluorescent Proteins; D013713:Teniposide; D004250:DNA Topoisomerases, Type II