Partitioning of peptides and recombinant protein-peptide fusions in thermoseparating aqueous two-phase systems: effect of peptide primary structure

Genetic engineering has been used for fusion of peptides, with different le ngth and composition, on a protein to study the effect on partitioning in a n aqueous two-phase system. The system was composed of dextran and the ther moseparating ethylene oxide-propylene oxide random copolymer, EO30PO70. Pep tides containing tryptophan, proline, arginine or aspartate residues were f used at the C-terminus of the recombinant protein ZZ-cutinase. The aim was to find effective tags for the lipolytic enzyme cutinase for large-scale ex traction. The target protein and peptide tags were partitioned separately a nd then together in the fusion proteins in order to gain increased understa nding of the influence of certain amino acid residues on the partitioning. The salt K2SO4 was used to reduce the charge dependent salt effects on part itioning and to evaluate the contribution to the partition coefficient from the hydrophobic-hydrophilic properties of the amino acid residues. The eff ect of Trp on peptide partitioning was independent of the difference in pri mary structure for (Trp)n, (Trp-Pro)n, (Ala-Trp-Trp-Pro)n and was only dete rmined by the number of Trp. The effect of the charged residues, Arg and As p, was dependent on the surrounding residues, i.e. if they were situated ne xt to Trp or not. The partitioning behaviour observed for the peptides was qualitatively and in some cases also quantitatively the same as for the fus ion proteins. The effect of the salts sodium perchlorate and triethylammoni um phosphate on the partitioning was also studied. The salt effects observe d for the peptides were qualitatively similar to the effects observed for t he fusion proteins. (C) 2000 Elsevier Science B.V. All rights reserved.