Chemical treatment of Escherichia coli: 3. Selective extraction of a recombinant protein from cytoplasmic inclusion bodies in intact cells

In previous parts of this study we developed procedures for the high-effici ency chemical extraction of soluble and insoluble protein from intact Esche richia coli cells. Although high yields were obtained, extraction of recomb inant protein directly from cytoplasmic inclusion bodies led to low product purity due to coextraction of soluble contaminants. In this work, a two-st age procedure for the selective extraction of recombinant protein at high e fficiency and high purity is reported. In the first stage, inclusion-body s tability is promoted by the addition of 15 mM 2-hydroxyethyldisulfide (2-HE DS), also known as oxidized P-mercaptoethanol, to the permeabil ization buf fer (6 M urea + 3 mM ethylenediaminetetra-acetate [EDTA]). 2-HEDS is an oxi dizing agent believed to promote disulfide bond formation, rendering the in clusion body resistant to solubilization in 6 M urea. Contaminating protein s are separated from the inclusion-body fraction by centrifugation. in the second stage, disulfide bonds are readily eliminated by including reducing agent (20 mM dithiothreitol [DTT]) into the permeabilization buffer. Extrac tion using this selective two-stage process yielded an 81% (w/w) recovery o f the recombinant protein Long-R-3-IGF-I from inclusion bodies located in t he cytoplasm of intact E. coli, at a purity of 46% (w/w). This was comparab le to that achieved by conventional extraction (mechanical disruption follo wed by centrifugation and solubilization). A pilot-scale procedure was also demonstrated using a stirred reactor and diafiltration. This is the first reported study that achieves both high extraction efficiency and selectivit y by the chemical treatment of cytoplasmic inclusion bodies in intact bacte rial cells. (C) 1999 John Wiley & Sons, Inc.