Genetic engineering of protein-peptide fusions for control of protein partitioning in thermoseparating aqueous two-phase systems

Genetic engineering has been used for the fusion of peptides, with differen t length and composition, on a protein to study the effect on partitioning in aqueous two-phase systems containing thermoseparating polymers. Peptides containing 2-6 tryptophan residues or tryptophan plus 1-3 lysine or aspart ate residues, were fused near the C-terminus of the recombinant protein ZZT 0, where Z is a synthetic IgG-binding domain derived from domain B in staph ylococcal protein A. The partitioning behavior of the peptides and fusion p roteins were studied in an aqueous two-phase system composed of dextran and the thermoseparating ethylene oxide-propylene oxide random copolymer, EO30 PO70. The zwitterionic compound p-alanine was used to reduce the charge-dep endent salt effects on partitioning, and to evaluate the contribution to th e partition coefficient from the amino acid residues, Trp, Lys, and Asp, re spectively. Trp was found to direct the fusion proteins to the EO-PO copoly mer phase, while Asp and Lys directed them to the dextran phase. The effect of sodium perchlorate and triethylammonium phosphate on the partitioning o f the fusion proteins was also studied. Salt effects were directly proporti onal to the net charge of the fusion proteins. Sodium perchlorate was found to be 3.5 times more effective in directing positively charged proteins to the EO-PO copolymer phase compared to the effect of triethyl ammonium phos phate on negatively charged proteins. An empirical correlation has been tes ted where the fusion protein partitioning is a result of independent contri butions from unmodified protein, fused peptide, and salt effects. A good ag reement with experimental data was obtained which indicates the possibility , by independent measurements of partitioning of target protein and fusion peptide, to approximately predict the fusion protein partitioning. (C) 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 135-144, 1999.