Optimization of an osmotic shock procedure for isolation of a protein product expressed in E. coli.
Small-scale experiments were performed to evaluate the impact of several processing variables on recovery by osmotic shock of a protein product expressed in E. coli utilizing feed material manufactured at large scale. Equilibration time, type and concentration of osmotic supplements, and pH of the osmotic shock buffer were found to have significant impact on the osmotic shock recovery. Improved recovery was documented under conditions of increased equilibration time before the osmotic shock and increased Tris-HCl concentration in the osmotic shock buffer, as well as initial dispersal of cells into the osmotic shock buffer. Recovery of up to 65% was achieved under the optimized conditions.
Rathore
Anurag S
AS
Bioprocess and Formulation Technology, Pharmacia Corporation, 700 Chesterfield Parkway North, Chesterfield, Missouri 63198, USA. arathore@amgen.com
Bilbrey
Robert E
RE
Steinmeyer
David E
DE
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D002458:Cell Fractionation / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D019382:Human Growth Hormone / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D009997:Osmotic Pressure; D011786:Quality Control; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013395:Sucrose / Q000378:metabolism*; D013696:Temperature
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D019382:Human Growth Hormone; D013395:Sucrose