Aqueous two-phase systems containing urea: influence on phase separation and stabilization of protein conformation by phase components.
During recombinant Escherichia coli fermentation with high expression levels, inclusion bodies are often formed. Aqueous two-phase systems have been used in the presence of urea for the initial recovery steps. To investigate phase behavior of such systems we determined phase diagrams of poly(ethylene glycol) (PEG)/sodium sulfate/urea/water and PEG/dextran T-500 (DEX)/urea/phosphate buffer/water at different concentrations of urea and different molecular weight of PEG. PEG/Na2SO4 aqueous two-phase systems could be obtained including up to 30% w/w urea at 25 degrees C and PEG/dextran T-500 up to 35% w/w urea. The binodial was displaced toward higher concentrations with increasing urea concentrations. The partition coefficient of urea was near unity. An unstable mutant of T4-lysozyme with an amino acid replacement in the core (V149T) was used to analyze the effect of phase components on the conformation of the enzyme. We showed that partitioning of tryptophan was not dependent on the concentration of urea in the phase system.
Rämsch
C
C
Institute of Enzymetechnology, Heinrich-Heine Universität Düsseldorf, D-52428 Jülich, Germany.
Kleinelanghorst
L B
LB
Knieps
E A
EA
Thömmes
J
J
Kula
M R
MR
D019943:Amino Acid Substitution; D001709:Biotechnology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D002479:Inclusion Bodies / Q000378:metabolism; D009113:Muramidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D014364:Tryptophan; D014508:Urea; D014867:Water
D011994:Recombinant Proteins; D014867:Water; D014364:Tryptophan; D014508:Urea; D009113:Muramidase