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Chemical treatment of Escherichia coli. II. Direct extraction of recombinant protein from cytoplasmic inclusion bodies in intact cells
Author(s) -
Falconer Robert J.,
O'Neill Brian K.,
Middelberg Anton P. J.
Publication year - 1998
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19980220)57:4<381::aid-bit1>3.0.co;2-i
Subject(s) - dithiothreitol , inclusion bodies , recombinant dna , extraction (chemistry) , chemistry , cytoplasm , escherichia coli , chromatography , cell disruption , urea , dissolution , protein purification , biochemistry , biophysics , biology , enzyme , organic chemistry , gene
A method is presented for the direct extraction of the recombinant protein Long‐R 3 ‐IGF‐I from inclusion bodies located in the cytoplasm of intact Escherichia coli cells. Chemical treatment with 6 M urea, 3 m M EDTA, and 20 m M dithiothreitol (DTT) at pH 9.0 proved an effective combination for extracting recombinant protein from intact cells. Comparable levels of Long‐R 3 ‐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‐R 3 ‐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. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57:381‐386, 1998.