Chemical treatment of Escherichia coli: 1. Extraction of intracellular protein from uninduced cells

• Published in: Biotechnology and bioengineering 1997-03, Vol.53 (5), p.453-458
• Main Authors: Falconer, Robert J., O'Neill, Brian K., Middelberg, Anton P. J.
• Format: Article
• Language: English
• Subjects: Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Biotechnology; cell disruption; chemical permeabilization; EDTA; Escherichia coli; Fundamental and applied biological sciences. Psychology; Methods. Procedures. Technologies; Microbiology; Others; urea; Various methods and equipments
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/(SICI)1097-0290(19970305)53:5<453::AID-BIT2>3.0.CO;2-G

Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical disruption. A chemical treatment that destroys the integrity of the bacterial cell wall and could provide an alternative technique is examined in this study. Treatment with a combination of the chelating agent ethylenediaminetet‐raacetate (EDTA) (greater 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 presence of 3 mM EDTA can release cytoplasmic protein from both logarithmic‐phase and stationary‐phase E. coli cells at levels equivalent to mechanical 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 kinetics 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 urea concentration. Kinetics for 3 mM EDTA without urea is considerably slower, with a time constant of 12.3 min. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 453–458, 1997.