High pressure homogenization and the separation of soluble protein from recombinant Escherichia coli lysate using crossflow membrane filtration

Recombinant Escherichia coli often accumulates the recombinant protein of interest as insoluble inclusion bodies. Removing the soluble proteins from the inclusion bodies following cell disruption is a difficult processing step. Pretreatment of recombinant E. coli with 1.5 M guanidine HCl and 1.5% Triton X-100 allowed for reductions in the number of passes required for cell disruption and in the number of downstream processing steps required for the recovery of protein from the inclusion bodies. The combination of guanidine HCl and Triton X-100 gave higher permeate fluxes than guanidine HCl alone during crossflow membrane filtration of E. coli cell lysates. Low concentrations of urea had less of an effect on permeate flux and protein transmission than cell lysate solutions containing guanidine HCl. Increasing cell disruption had no effect on permeate flux and protein transmission because of the release of macromolecules and small cell debris after one pass through the high pressure homogenizer. A constant volume diafiltration process was developed to remove 84% of the UV...