EFFECTS OF FUSED TRYPTOPHAN RICH PEPTIDES TO A RECOMBINANT PROTEIN-A DOMAIN ON THE PARTITIONING IN POLYETHYLENE GLYCOL-DEXTRAN AND UCON-DEXTRAN AQUEOUS 2-PHASE SYSTEMS

Genetic engineering has been used to construct fusion proteins with tr yptophan containing peptides. The peptides and the fusion proteins hav e been partitioned in aqueous two-phase systems of poly(ethylene glyco l) (PEG)-dextran and Ucon-dextran. The studied model protein was ZZT0, where Z is an engineered domain of domain B of staphylococcal protein A. The specially designed hydrophobic peptides, Ala-Trp-Trp-Pro (T1) and (Ala-Trp-Trp-Pro), (T2), have been inserted into ZZT0, to give the peptide-protein fusions ZZT1 and ZZT2. In the experimental studies it was found that T1 and T2 preferred the PEG phase and even more the Uc on phase over the dextran phase. For T2 the partitioning was more one sided than for T1. For the fusion proteins, ZZT1 and ZZT2, the partiti oning was enhanced into the PEG or Ucon rich phase as compared to ZZT0 . The effects were lower than expected from independent contributions to the partition coefficient from the protein and the peptides. A hete rogeneous lattice model was used to calculate theoretical peptide and protein partition coefficients. The calculations could reproduce the q ualitative features of the experimental data. The model results sugges t that a part of these experimentally observed effects is due to a dep letion zone, i.e. a zone of reduced polymer concentration around the p rotein. The experimental results indicate a further reduction of the p artition coefficient, beyond that predicted by the lattice calculation s. A possible folding of the inserted peptide is discussed as a plausi ble mechanism for this further reduction in the partition coefficient.