Aqueous Two-Phase Systems Containing Urea: Influence on Phase Separation and Stabilization of Protein Conformation by Phase Components

• Published in: Biotechnology progress Vol. 15; no. 3; pp. 493 - 499
• Main Authors: Rämsch, Christian, Kleinelanghorst, Lutz B., Knieps, Esther A., Thömmes, Jörg, Kula, Maria-Regina
• Format: Journal Article
• Language: English
• Published: USA American Chemical Society 1999; American Institute of Chemical Engineers
• Subjects: Amino Acid Substitution; Amino acids; Biological and medical sciences; Biotechnology; Conformations; Enzyme Stability; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fermentation; Fundamental and applied biological sciences. Psychology; Inclusion Bodies - metabolism; Methods. Procedures. Technologies; Muramidase - chemistry; Muramidase - genetics; Muramidase - isolation & purification; Others; Phase composition; Phase diagrams; Phase separation; Point Mutation; Polyethylene glycols; Protein Conformation; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Tryptophan; Urea; Various methods and equipments; Water; Enzyme; Escherichia coli; Tryptophan; Ethylene oxide polymer; Separation method; Urea; Biphasic system; Aqueous medium; Glycosidases; Bacteria; Hydrolases; Phase partition; Inclusion body; Recombinant protein; O-Glycosidases; Lysozyme; Enterobacteriaceae; Conformation
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1021/bp990030+

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 °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.