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        Overexpression and Purification of Saccharomyces cerevisiae DNA Topoisomerase II from Yeast

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        Mechanistic and structural studies require large quantities of highly purified enzyme. Unfortunately, traditional expression strategies using Escherichia coli are often not successful for eukaryotic proteins, especially large ones. Despite numerous attempts, overexpression and purification of Saccharomyces cerevisiae DNA topoisomerase II from E. coli proved unsuccessful (Worland and Wang, personal communication). This failure may be owing to the common occurrence of the rare E. coli codons CTA (leucine) and AGG (arginine) in the yeast gene, particularly in the carboxy-terminal half. In response, Worland and Wang developed an expression and purification system for topoisomerase II in yeast (1 ). This procedure and its variations have been immensely useful; 5 mg of wild-type or mutant topoisomerase II can generally be purified from 1 L of cells grown in selective media. This chapter describes methods for growing and inducing the yeast cells, and purifying the highly expressed type II DNA topoisomerase. Researchers who are unfamiliar with yeast may find several chapters in ref. (2 ) useful.
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