IMPROVED HOMOGENIZATION OF RECOMBINANT ESCHERICHIA-COLI FOLLOWING PRETREATMENT WITH GUANIDINE-HYDROCHLORIDE

Pretreatment of recombinant Escherichia coli, expressing human growth hormone inclusion bodies, with guanidine hydrochloride and Triton X-10 0 prior to high-pressure homogenization has been investigated. Homogen ates were analyzed for protein release, viscosity, and particle size. We were able to reduce the number of passes required for cell disrupti on and the number of downstream processing steps required for the reco very of protein from inclusion bodies by pretreating cells with guanid ine HCl and Triton X-100. Pretreatment of exponential growth phase cel ls with 1.5 M guanidine HCl and 1.5% Triton X-100 gave adequate disrup tion after one pass at 41 MPa with a particle size distribution simila r to that for untreated cells disrupted after one pass at 62 MPa. This combination of guanidine HCl and Triton X-100 was also selected so as to wash the inclusion bodies without solubilization of the human grow th hormone. Pretreatment of cells with 4 M guanidine HCl produced cell debris that was substantially smaller than the debris from untreated cells and partially solubilized the inclusion bodies. Cells harvested in the stationary growth phase were more resistant to high-pressure ho mogenization and pretreatment.