Aqueous two-phase systems containing urea: Influence of protein structure on protein partitioning

During recombinant E. coli fermentation with high-expression levels inclusi on bodies are often formed. Aqueous two-phase systems have been successfull y used in the presence of urea for the initial recovery step of inclusion b odies from E, coli. Basic studies of the complex interactions are lacking. For a systematic study of protein partitioning in the presence of urea we s elected T4-lysozyme mutants with different thermal stability as a model. Th e stabilization of these variants by phase components was investigated meas uring the fluorescence emission of tryptophan residues in the protein. Prot ein structure was stabilized at pH 7 in the order of SO42- >> PEG = Dextran > H2O. The conformation of proteins was shown to have a strong influence o n the partitioning in aqueous two-phase systems. Tryptophan and its homolog uous di- and tripeptdides were partitioned in similar phase systems to norm alize for contribution from hydrophobic interactions. (C) 2000 John Wiley & Sons, Inc.