50] Proteases in Escherichia coli

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Publisher Summary This chapter presents the procedure for purification and assaying of proteases in Escherichia coli. The purification procedure involves preparation of cell-free extract and fractionation of cell-free extract on DEAE-cellulose. For preparation of cell-free extract, freshly grown, or frozen E. coli K12 cells are suspended in buffer. The cell suspension is disrupted in a French press at 14,000 psi, the resulting homogenate is centrifuged at 31,000 g for 30 min, and the supernatant at 130,000 g for 3 hr. Following extensive dialysis against buffer B, the insoluble material is removed by centrifugation. In fractionation dialyzed cell-free extract is applied to a DEAEcellulose column equilibrated with buffer. The column is washed with the same buffer until the Azso of the eluate is less than 0.05. The adsorbed proteins are then eluted with 1500 ml of a linear salt gradient. Alternate fractions are assayed for proteolytic activity against [3H]casein and [125I]insulin in the presence and absence of 3 mM ATP. Different peaks of proteolytic activity against these substrates are found. For convenience, they are designated peaks I-V, according to their order of elution from the column. To obtain sufficient material for further purification, peaks from several DEAE-cellulose columns can be pooled. The following steps are described for enzyme preparations that would correspond to starting with 250-300 g of frozen cells. For various proteases in different peak individual chromatograpgy is carried out for respective proteases.