Peptide-tagged proteins in aqueous two-phase systems

Abstract: This thesis deals with proteins containing peptide tags for improved partitioning in aqueous two-phase systems. Qualitatively the peptide-tagged protein partitioning could be predicted from peptide data, i.e. partitioning trends found for peptides were also found for the peptide-tagged proteins. However, full effect of the tag as expected from peptide partitioning was not found in the tagged protein. When alkyl-ethylene oxide surfactant was included in a two-polymer system, almost full effect of the tag was obtained. This indicates an improved exposure of the tag to the phase components in the surfactant-containing systems. The most efficient amino acid residue for partitioning towards a phase rich in random copolymer of ethylene oxide and propylene oxide (EOPO) was tryptophan. The second most efficient amino acids were tyrosine and phenylalanine. The tag efficiencies were higher for proteins with tyrosine/proline tags compared to proteins with tryptophan/proline tags. The reason can be that the tyrosine residues are more exposed to the solution and phase-forming components, since tyrosine is more hydrophilic than tryptophan. The partitioning coefficient of cutinase wild-type was calculated from peptide partitioning data combined with surface studies with the computer program GRASP. The calculated partitioning coefficient agreed relatively well with the experimentally determined partition coefficient. Thus, it is possible to obtain an approximate partitioning coefficient of a protein before starting a purification procedure and thereby save time in finding an optimal partitioning system. The fluorescence emission maximum wavelength for free peptide was longer than for peptide tag in tryptophan-tagged cutinase, indicating larger exposure of free peptides. However, all maxima were obtained at wavelengths corresponding to a polar environment and thus indicating solvent exposure of the tryptophan residues in both free peptides and tags. The emission maximum of the tryptophan tag was moved to longer wavelength when a spacer was introduced between protein and tag. This correlates with results obtained in aqueous two-phase partitioning where a spacer between tag and protein resulted in increased partitioning towards an EOPO copolymer phase.