High purity, catalytically active Human Topoisomerase I Enzyme over-expressed in the baculovirus system and purified to single band homogeneity. These preparations are cheaper than native Topo I 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.
Materials Safety Data
Quickstart Guide for Topo I
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