AbstractsBiology & Animal Science

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. När en dressing tillreds används ofta olja, vinäger och kryddor. Även om dressingen skakas ordentligt kommer den efter ett tag att dela upp sig i två lösningar, en med olja och en med vinäger. Ett tvåfassystem har bildats där kryddorna fördelar sig mellan olje- och vinägerfasen. Inom biokemiforskning används tvåfassystem av vatten och polymerer. Polymerer är stora molekyler som består av mängder av små enheter, som är sammankopplade till långa kedjor. Genom att blanda två olika sorters polymerer med vatten, kan vattnet på liknande sätt som oljan och vinägern i dressingen dela upp sig i två faser. Polymererna löser sig i vattnet, men lösningen separerar i två faser med i stort sett bara en typ av polymer i varje fas. På motsvarande sätt som kryddorna i dressingen…