[1] Purification of Escherichia coli DNA polymerase I and Klenow fragment

This chapter describes the most recent constructs and protocols, which typically yields of 10 mg of pure polymerase per gram of cells. DNA polymerase I (Pol I) of Escherichia coli (E. coli), the first DNA polymerase to be discovered, has long served as a simple model system for studying the enzymology of DNA synthesis. The original studies of Pol I relied on purification of the enzyme from E. coli extracts without genetic manipulation, yielding around 10 mg of purified enzyme per kilogram of cell paste. The ability to purify large quantities of Klenow fragment paved the way for the determination of its structure by X-ray crystallography. Both Pol I and Klenow fragment have found extensive use as biochemical reagents in a variety of cloning, sequencing, and labeling procedures. The procedure described for the purification of Escherichia coli DNA polymerase I and Klenow fragment makes use of the Pharmacia fast protein liquid chromatography (FPLC) system. If this equipment is not available, published procedures using conventional chromatography are satisfactory.