Peptide-tagged proteins in aqueous two-phase systems

Research output: ThesisDoctoral Thesis (compilation)

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.

Details

Authors
  • Anna Nilsson
Organisations
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Biological Sciences

Keywords

  • Metabolism, Biokemi, metabolism, Biochemistry, green fluorescent protein, cutinase, tryptophan, Aqueous two-phase system, peptide-tagged protein
Original languageEnglish
QualificationDoctor
Awarding Institution
Supervisors/Assistant supervisor
  • [unknown], [unknown], Supervisor, External person
Award date2002 Oct 25
Publisher
  • Prof. Folke Tjerneld, Dept. Biochemistry, Lund University
Print ISBNs91-7422-009-8
Publication statusPublished - 2002
Publication categoryResearch

Bibliographic note

Defence details Date: 2002-10-25 Time: 10:15 Place: Lecture Hall B, Chemical Centre External reviewer(s) Name: Van Alstine, James Title: Prof Affiliation: Amersham Biosciences, Uppsala --- Article: I. Partitioning of peptides and recombinant protein-peptide fusions in thermoseparating aqueous two-phase systems: effect of peptide primary structureKristina Berggren, Anna Nilsson, Göte Johansson, Nina Bandmann, Per-Åke Nygren, Folke TjerneldJournal of Chromatography B 743 (2000) 295-306 Article: II. Cutinase-peptide fusions in thermoseparating aqueous two-phase systems. Prediction of partitioning and enhanced tag efficiency by detergent additionAnna Nilsson, Maurice Mannesse, Maarten R. Egmond, Folke TjerneldJournal of Chromatography A 946 (2002) 141-155 Article: III. Partitioning of peptide-tagged proteins in aqueous two-phase systems using hydrophobically modified micelle-forming thermoseparating polymerAnna Nilsson, Hans-Olof Johansson, Maurice Mannesse, Maarten R. Egmond, Folke TjerneldBiochimica et Biophysica Acta, Accepted Article: IV. Tryptophan-tagged cutinase studied by steady-state fluorescence for understanding of tag interactions in aqueous two-phase systemsAnna Nilsson, Maria Teresa Neves-Petersen, Hans-Olof Johansson, Jörgen Jansson, Karin Schillén, Folke Tjerneld, Steffen B. PetersenSubmitted Article: V. Partitioning and characterisation of tyrosine-tagged green fluorescent proteins in aqueous two-phase systemsSara Fexby, Anna Nilsson, Gustav Hambraeus, Folke Tjerneld, Leif BülowSubmitted

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