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.

During recombinant E. coli fermentation with high-expression levels inclusion bodies are often formed. Aqueous two-phase systems have been successfully used in the presence of urea for the initial recovery step of inclusion bodies from E. coli. Basic studies of the complex interactions are lacking. For a systematic study of protein partitioning in the presence of urea we selected T4-lysozyme mutants with different thermal stability as a model. The stabilization of these variants by phase components was investigated measuring the fluorescence emission of tryptophan residues in the protein. Protein structure was stabilized at pH 7 in the order of S0(4)(2-) >> PEG = Dextran > H(2)O. The conformation of proteins was shown to have a strong influence on the partitioning in aqueous two-phase systems. Tryptophan and its homologuous di- and tripeptdides were partitioned in similar phase systems to normalize for contribution from hydrophobic interactions.

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