In order to carry out studies on the structure and mechanism of enzymes, substantial quantities of purified proteins are often needed for many of the commonly used biophysical methods. This is especially true for three-dimensional structure determination using X-ray crystallography or NMR. Structure–function analysis by site-directed mutagenesis requires that a large number of mutant enzymes be expressed and purified readily, so that their properties can be compared to those of the purified wild-type enzyme. Biophysical characterizations of a mutant enzymes are desirable to assess if the mutation has altered the folded conformation of the enzyme. Therefore, it is necessary to overexpress the protein of interest to maximize the yield and facilitate the purification process. For these reasons, Escherichia coli DNA topoisomerase I and several of its partial fragments have been purified previously after overexpression (1-5). The methods involved should in general be applicable for overexpression and purification of bacterial topoisomerase I.