Human Topo I Product Background
High purity, catalytically active Top1 Enzyme overexpressed 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.
Quality Control Tests:
- A test for nuclease contaxamination was carried out by assaying for the formation of linear KDNA and linear plasmid DNA. Incubations of 1 µg of catenated KDNA or supercoiled pUC19 DNA (4 hrs. at 37°C in the presence of 10 mM MgCl2) were performed. Linear DNA or breakdown products were not generated under these conditions.
- A check for cross contamination with topo II was negative. There was no decatenation of kDNA in topo II reaction conditions.
- The final fraction of topoisomerase 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.
*Actual concentration can range from 25mM-100mM, according to how stock enzyme was diluted in storage buffer.
Relaxation assays were carried out in a final volume of 25 µl in recombinant topo I reaction buffer (10X reaction buffer, supplied with this product is: 100 mM Tris-Cl, pH 7.9, 1.5 M NaCl, 1% BSA, 1 mM Spermidine, 50% glycerol). Supercoiled plasmid DNA was included at 0.25 µg/reaction. Reactions terminated with 5 µl (per 20 µl reaction volume) of stop buffer (5% sarkosyl, 0.0025% bromophenol blue, 25% glycerol). Reaction products were analyzed on a 1% native agarose gel. Under these conditions, relaxation activity is easily detectable (typically we see 10-40 units of activity per ul). A typical titration is shown below:
- 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
10X assay buffer and dilution buffer is included. These may be purchased separately as TGS 10X Topo I Assay Buffer (TG4095).
The 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.