Chemical treatment of Escherichia coli. II. Direct extraction of recombinant protein from cytoplasmic inclusion bodies in intact cells.
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.
Falconer
R J
RJ
Cooperative Research Centre for Tissue Growth and Repair, Department of Chemical Engineering, The University of Adelaide, SA 5005, Australia.
O'Neill
B K
BK
Middelberg
A P
AP
D001671:Biochemistry / Q000379:methods*; D002463:Cell Membrane Permeability; D004229:Dithiothreitol / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry* / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000031:analogs & derivatives / Q000302:isolation & purification; D007700:Kinetics; D011994:Recombinant Proteins / Q000302:isolation & purification*; D014508:Urea / Q000737:chemistry
D011994:Recombinant Proteins; C085700:long R(3)-insulin-like growth factor-I; D007334:Insulin-Like Growth Factor I; D014508:Urea; D004492:Edetic Acid; D004229:Dithiothreitol