Mutant Human Topoisomerase I


10 ug

SKU: TG2005H-Y723F Category:


Mutant Human Topoisomerase I Product Description
TopoGEN now offers a catalytically inactive form of mutant 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
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° 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 mutant topo 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 (see below).

Topo I Mutated Baculovirus Data

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.

Shipping and Storage Instructions:

The enzyme is shipped on blue ice or dry ice internationally. Store the enzyme at -20°C.