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-100 prior to high-pressure homogenization has been investigated. Homogenates were analyzed for protein release, viscosity, and particle size. We were able to reduce the number of passes required for cell disruption and the number of downstream processing steps required for the recovery of protein from inclusion bodies by pretreating cells with guanidine HCl and Triton X-100. Pretreatment of exponential growth phase cells with 1.5 M guanidine HCl and 1.5% Triton X-100 gave adequate disruption after one pass at 41 MPa with a particle size distribution similar 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 growth 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 homogenization and pretreatment.
Department of Biological Systems Engineering, School of Biological Sciences, University of Nebraska, Lincoln 68583-0919, USA.
S M
SM
Bailey
P H
PH
Blum
M M
MM
Meagher
D001426:Bacterial Proteins / Q000187:drug effects* / Q000302:isolation & purification*; D001709:Biotechnology / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry* / Q000187:drug effects* / Q000378:metabolism; D005285:Fermentation; D013006:Growth Hormone / Q000096:biosynthesis / Q000302:isolation & purification; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry*; D017830:Octoxynol / Q000494:pharmacology; D010316:Particle Size; D011994:Recombinant Proteins / Q000187:drug effects / Q000302:isolation & purification; D014783:Viscosity
D001426:Bacterial Proteins; D006146:Guanidines; D011994:Recombinant Proteins; D013006:Growth Hormone; D017830:Octoxynol; D019791:Guanidine