CHEMICAL TREATMENT OF ESCHERICHIA-COLI .1. EXTRACTION OF INTRACELLULAR PROTEIN FROM UNINDUCED CELLS

Extraction of intracellular protein from Escherichia coli is tradition ally achieved by mechanical disruption. A chemical treatment that dest roys the integrity of the bacterial cell wall and could provide an alt ernative technique is examined in this study. Treatment with a combina tion of the chelating agent ethylenediaminetetraacetate (EDTA) (greate r than 0.3 mM) and the chaotropic agent urea (6 M) is highly effective at releasing protein from uninduced E. coli. The 6 M urea in the pres ence of 3 mM EDTA can release cytoplasmic protein from both logarithmi c-phase and stationary-phase E. coli cells at levels equivalent to mec hanical disruption. The concentrations of the two chemical agents were the major variables affecting the maximum levels of protein release. Several minor variables and interactions were also identified. The kin etics of protein release is first order. For 2, 4, and 6 M urea with 3 mM EDTA, the time constant is approximately 2.5 min independent of ur ea concentration. Kinetics for 3 mM EDTA without urea is considerably slower, with a time constant of 12.3 min. (C) 1997 John Wiley & Sons, Inc.