Protein Release from Escherichia Coli Cells Permeabilized with Guanidine-Hcl and Triton X100.

Abstract

An important factor complicating the recovery of recombinant protein products from E. coli is their intracellular location. Chemical treatment of the cells with guanidine-HCl and Triton X100 has been shown to be a feasible alternative to mechanically based cell disruption methods. Some of the advantages of chemical treatment with guanidine-HCl and Triton X100 include avoidance of cell fragmentation, retention of the nucleic acids inside the cell, and ease of operation. The mechanism by which guanidine and Triton permeabilize E. coli to protein was found to include extensive solubilization of the inner membrane and no apparent alteration of the peptidoglycan layer and outer membrane. Guanidine was capable of causing significant disruption of the inner membrane into fragments and vesicles whereas Triton was effective in solubilizing these fragments and vesicles. The alteration of the outer wall was thought to be on the molecular level and may include solubilization of some of the outer membrane lipids and proteins. Variation of the guanidine and Triton concentrations was found to lead to a set of protein release processes with different kinetic properties. These kinetic properties, i.e. the protein release rate and yield, were mapped out for guanidine concentrations up to 4M and Triton concentrations up to 2%. The protein release processes did not give a selective release of protein nor was the yield found to depend on osmotic shock or a depletion of the permeabilizing chemicals. In certain concentration regions, the cells displayed a distribution of responses. These cell-to-cell differences appear to be related to a distribution of cellular physiological states. A complex dependence of the release kinetics on guanidine was observed for treatments with a constant Triton concentration between .5% and 2%. As the guanidine concentration was increased to 4M, the release rate varied monotonically but the yield varied in a wave-like manner. This complex dependence on guanidine resulted from an interaction between two opposing phenomena: protein release from the cells and protein aggregation inside the cell. A single cell model incorporating these phenomena gave qualitative agreement to the experimental data. The model used an average cell with a time invariant permeability coefficient to represent the cell population.