Mutant Human Topo I

Catalog#	Description	Size
2005H-Y723F	Mutant Human Topo I (Y723F)	10ug

Description:

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TopoGEN now offers a catalytically inactive mutant form of human topoisomerase I purified to homogeneity (single band on SDS-PAGE). The mutation is a single residue change at the active site tyrosine (which has been changed to phenylalanine). This preparation is overexpressed in baculovirus and affinity purified as a single band on SDS-PAGE of 100 kDa. The enzyme cannot relax DNA (but still binds to DNA) and contains a short (6 residue) histidine tag.

Quality Control Tests:

1. A test for nuclease contamination 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° in the presence of 10 mM MgCl2) were performed. Linear DNA or breakdown products were not generated under these conditions.

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 topoisomerase I is a column pool and is in the following buffer: 20mM NaH2PO4 (pH7.4), 300mM NaCl, 500mM Imidazole. The final fraction was analyzed by SDS-PAGE and shown to contain a single, predominant band of 100 kDa (Fig. 1 below).

Activity assays:

Relaxation assays were carried out in a final volume of 25 µl in 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 was not detectable with up to 0.5 ug of mutant protein.

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