Use of multifactorial analysis to develop aqueous two-phase systems for isolation of non-native IGF-I.
A high yield procedure was developed to solubilize and extract IGF-I from recombinant E. coli by adding chaotrope and disulfide reductant to alkaline fermentation broth. To enhance centrifugation performance and recovery yield, a salt/polymer aqueous two-phase extraction procedure was developed whereby soluble non-native IGF-I and biomass solids are enriched in separate liquid phases. To develop this extraction system a multifactorial experimental approach was used to simultaneously map the phase diagram and identify conditions to suitably partition IGF-I and cell remnants. The presence of urea in these systems tended to disrupt two-phase formation and solids sedimentation. This, in turn, constrained the concentrations of phase forming solutes which could be effectively used. Systems containing low levels of salt (less than about 4% w/w) and polymer (less than about 10% w/w) did not form two phases. Systems containing high levels of salt (greater than about 7% w/w) and polymer (greater than about 18% w/w) formed two phases with floating solids. Intermediate levels of salt (between about 4% and 7% w/w) and polymer (between about 10% and 18% w/w) formed two phases in which solids were enriched in the heavy phase. Systems in this latter desired category were produced with a variety of different salts and polymers and all enriched non-native IGF-I in the light phase. Highest recovery yield (about 90%) was obtained with systems composed of 5% sodium sulfate and 14% PEG-8000.(ABSTRACT TRUNCATED AT 250 WORDS)
Hart
R A
RA
Department of Recovery Sciences, Genentech Inc., South San Francisco, CA 94080-4918, USA.
Ogez
J R
JR
Builder
S E
SE
D002498:Centrifugation; D004926:Escherichia coli / Q000235:genetics; D007202:Indicators and Reagents; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000302:isolation & purification*; D011092:Polyethylene Glycols; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D013431:Sulfates; D014867:Water
D007202:Indicators and Reagents; D011994:Recombinant Proteins; D013431:Sulfates; D014867:Water; C012036:sodium sulfate; D011092:Polyethylene Glycols; D007334:Insulin-Like Growth Factor I