Recovery of a foreign protein from the periplasm of Escherichia coli by chemical permeabilization.
We have applied the technique of protein release by chemical permeabilization to recover a foreign protein in active form from the periplasm of a recombinant strain of Escherichia coli. The two agents used in our chemical permeabilization scheme, guanidine hydrochloride and Triton X-100, have different modes of action, allowing selectivity in protein release based on intracellular location under different treatment conditions. Specifically, treatment of E. coli C600-1 cells by guanidine alone resulted in 40-fold purification of recombinant beta-lactamase, which is periplasmically expressed in this host. Achieving such high purification in the cell disruption stage could alleviate some of the problems associated with recovery of intracellular products, such as low expression or the need to solubilize cytoplasmic inclusion bodies. Recovery of periplasmic proteins by chemical permeabilization is simpler than by osmotic shock and is less expensive than using enzymatic digestion.
Naglak
T J
TJ
Department of Chemical Engineering, University of Michigan, Ann Arbor 48109-2136.
Wang
H Y
HY
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D002463:Cell Membrane Permeability; D003902:Detergents; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D017830:Octoxynol; D011092:Polyethylene Glycols; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D003902:Detergents; D006146:Guanidines; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; D005954:Glucosephosphate Dehydrogenase; D001618:beta-Lactamases; D019791:Guanidine