High purity, catalytically active Human Topoisomerase I overexpressed in the baculovirus system and purified to single band homogeneity. These preparations are cheaper than native top1 made from human tissue and offer excellent purity and high specific activity. In addition, the 100 kDa form of recombinant topo I is stable and is less prone to proteolysis, relative to the native protein from human cells.
The final preparation of Topo 1 is highly active in relaxation of plasmid DNA.
Titration of Human Topoisomerase I Enzyme Activity
Topo I as prepared and purified using the baculovirus overexpression system. Activity was determined by serial 2 fold dilutions using supercoiled DNA. The data show that this preparation titrates to approximately 8 units/ul. One unit of Human Topo I can relax >50% of 0.25 μg (250 ng) of supercoiled plasmid DNA in 30 minutes at 37°C.
10X assay buffer and dilution buffer is included. These may be purchased separately as TGS 10X Topo I Assay Buffer (TG4095).
Quality Control Tests
1. Human Topo I is certified to be nuclease free.
2. A check for cross contamination with topo II was negative. There was no decatenation of KDNA in topo II reaction conditions.
3. The final fraction of topo I is a column pool and is in the following buffer: 20mM NaH2PO4 (pH7.4), 300mM NaCl, 50ug/ml BSA, 50% glycerol, 50mM Imidazole. The final fraction was analyzed by SDS-PAGE and shown to contain a single, predominant band of 100 kDa.
4. Activity assays. Relaxation assays were carried out in a final volume of 25 μl in top1 reaction buffer (10X reaction buffer–also called TGS–supplied with this product is: 100 mM Tris-Cl (pH 7.9), 10 mM EDTA, 1.5 M NaCl, 1% BSA, 1 mM Spermidine, 50% glycerol). Supercoiled plasmid DNA was included at 0.25 μg/reaction. Reactions are terminated with 5 μl (per 20 μl reaction volume) of stop buffer (5% sarkosyl, 0.0025% bromophenol blue, 25% glycerol). Reaction products are analyzed on a 1% native agarose gel (no ethidium bromide in the gel or buffer during electrophoresis).
Human Topoisomerase I enzyme is shipped on dry ice and should be stored at -20°C. It has a life-time of about 6 months when stored under optimal conditions. Repeat freeze/thaw cycles will accelerate loss.
Reviews and Citations
Yoshizaki A, Yanaba K, Ogawa A, Asano Y, Kadono T, Sato S: Immunization With DNA Topoisomerase I and Freund’s Complete Adjuvant Induces Skin and Lung Fibrosis and Autoimmunity via Interleukin-6 Signaling.Arthritis and Rheumatism 2011, 63: 3575-3585. doi: 10.1002/art.30539.
Zhang L, Sullivan P, Suyama J, et. al: Epidermal growth factor-induced heparanase nucleolar localization augments DNA topoisomerase I activity in brain metastatic breast cancer.Molecular Cancer Research 2010, 8: 278-290. doi: 10.1158/1541-7786.MCR-09-0375