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Titolo:
Chemical treatment of Escherichia coli: 3. Selective extraction of a recombinant protein from cytoplasmic inclusion bodies in intact cells
Autore:
Falconer, RJ; ONeill, BK; Middelberg, APJ;
Indirizzi:
Univ Cambridge, Dept Chem Engn, Cambridge CB2 3RA, England Univ CambridgeCambridge England CB2 3RA ngn, Cambridge CB2 3RA, England Univ Adelaide, Dept Chem Engn, Adelaide, SA 5005, Australia Univ AdelaideAdelaide SA Australia 5005 gn, Adelaide, SA 5005, Australia
Titolo Testata:
BIOTECHNOLOGY AND BIOENGINEERING
fascicolo: 4, volume: 62, anno: 1999,
pagine: 455 - 460
SICI:
0006-3592(19990220)62:4<455:CTOEC3>2.0.ZU;2-V
Fonte:
ISI
Lingua:
ENG
Keywords:
Escherichia coli; inclusion bodies; insulin-like growth factor; 2-hydroxyethyldisulfide;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Agriculture,Biology & Environmental Sciences
Life Sciences
Citazioni:
10
Recensione:
Indirizzi per estratti:
Indirizzo: Middelberg, APJ Univ Cambridge, Dept Chem Engn, Pembroke St, Cambridge CB23RA, England Univ Cambridge Pembroke St Cambridge England CB2 3RA land
Citazione:
R.J. Falconer et al., "Chemical treatment of Escherichia coli: 3. Selective extraction of a recombinant protein from cytoplasmic inclusion bodies in intact cells", BIOTECH BIO, 62(4), 1999, pp. 455-460

Abstract

In previous parts of this study we developed procedures for the high-efficiency chemical extraction of soluble and insoluble protein from intact Escherichia coli cells. Although high yields were obtained, extraction of recombinant protein directly from cytoplasmic inclusion bodies led to low product purity due to coextraction of soluble contaminants. In this work, a two-stage procedure for the selective extraction of recombinant protein at high efficiency and high purity is reported. In the first stage, inclusion-body stability is promoted by the addition of 15 mM 2-hydroxyethyldisulfide (2-HEDS), also known as oxidized P-mercaptoethanol, to the permeabil ization buffer (6 M urea + 3 mM ethylenediaminetetra-acetate [EDTA]). 2-HEDS is an oxidizing agent believed to promote disulfide bond formation, rendering the inclusion body resistant to solubilization in 6 M urea. Contaminating proteins are separated from the inclusion-body fraction by centrifugation. in thesecond stage, disulfide bonds are readily eliminated by including reducingagent (20 mM dithiothreitol [DTT]) into the permeabilization buffer. Extraction using this selective two-stage process yielded an 81% (w/w) recovery of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact E. coli, at a purity of 46% (w/w). This was comparable to that achieved by conventional extraction (mechanical disruption followed by centrifugation and solubilization). A pilot-scale procedure was also demonstrated using a stirred reactor and diafiltration. This is the firstreported study that achieves both high extraction efficiency and selectivity by the chemical treatment of cytoplasmic inclusion bodies in intact bacterial cells. (C) 1999 John Wiley & Sons, Inc.

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Documento generato il 02/07/20 alle ore 19:01:23