Please use this identifier to cite or link to this item:
Scopus Web of ScienceĀ® Altmetric
Type: Journal article
Title: Chemical Treatment of Escherichia coli. II Direct extraction of recombinant protein from cytoplasmic inclusion bodies in intact cells
Author: Falconer, R.
O'Neill, B.
Middelberg, A.
Citation: Biotechnology and Bioengineering, 1998; 57(4):381-386
Publisher: WILEY
Issue Date: 1998
ISSN: 0006-3592
Abstract: A method is presented for the direct extraction of the recombinant protein Long-R3-IGF-I from inclusion bodies located in the cytoplasm of intact Escherichia coli cells. Chemical treatment with 6M urea, 3 mM EDTA, and 20 mM dithiothreitol (DTT) at pH 9.0 proved an effective combination for extracting recombinant protein from intact cells. Comparable levels of Long-R3-IGF-I were recovered by direct extraction as achieved by in vitro dissolution following mechanical disruption. However, the purity of directly extracted recombinant protein was lower due to contamination by bacterial cell components. The kinetics of direct extraction are described using a first-order equation with the time constant of 3 min. Urea appears important for permeabilization of the cell and dissolution of the inclusion body. Conversely, EDTA is involved in permeabilization of the cell wall and DTT enhances protein release. pH proved to be important with lower levels of protein release achieved at low pH values (<9). Cell concentration also had a minor effect on Long-R3-IGF-I release and caused an observable increase in viscosity. Advantages of the direct extraction method include its speed, simplicity, and efficiency at releasing product.
Keywords: Inclusion Bodies
Escherichia coli
Edetic Acid
Insulin-Like Growth Factor I
Recombinant Proteins
Cell Membrane Permeability
Hydrogen-Ion Concentration
DOI: 10.1002/(SICI)1097-0290(19980220)57:4<381::AID-BIT1>3.0.CO;2-I
Appears in Collections:Aurora harvest 2
Chemical Engineering publications

Files in This Item:
There are no files associated with this item.

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.