Advanced

Tryptophan-tagged cutinase studied by steady state fluorescence for understanding of tag interactions in aqueous two-phase systems

Nilsson, Anna LU ; Neves-Petersen, Maria Teresa; Johansson, Hans-Olof LU ; Jansson, Jörgen LU ; Schillén, Karin LU ; Tjerneld, Folke LU and Petersen, Steffen B (2003) In Biochimica et Biophysica Acta - Proteins and Proteomics2002-01-01+01:00 1646(1-2). p.57-66
Abstract
Genetic engineering has been used to construct fusion proteins of Fusarium solani pisi cutinase and tryptophan-based tags, expressed in Saccharomyces cerevisiae, to increase the partitioning in aqueous two-phase systems. The separation systems were composed of thermoseparating polymers (random copolymers of ethylene oxide and propylene oxide, EOPO) and detergents (C12EOn). In this study, the fluorescence behaviour of the peptide-tagged protein, free peptide tag and tryptophan was investigated. The tryptophan-tagged proteins, cutinase-(WP)4 and cutinase-TGGSGG-(WP)4, showed emission spectra similar to the free peptides and tryptophan, indicating solvent exposure of the tag. The influence of polymers and detergents on the fluorescence of... (More)
Genetic engineering has been used to construct fusion proteins of Fusarium solani pisi cutinase and tryptophan-based tags, expressed in Saccharomyces cerevisiae, to increase the partitioning in aqueous two-phase systems. The separation systems were composed of thermoseparating polymers (random copolymers of ethylene oxide and propylene oxide, EOPO) and detergents (C12EOn). In this study, the fluorescence behaviour of the peptide-tagged protein, free peptide tag and tryptophan was investigated. The tryptophan-tagged proteins, cutinase-(WP)4 and cutinase-TGGSGG-(WP)4, showed emission spectra similar to the free peptides and tryptophan, indicating solvent exposure of the tag. The influence of polymers and detergents on the fluorescence of tagged proteins was examined. When peptides and tagged proteins were exposed to polymer, a slight blue shift of the emission maximum was observed. Larger blue shifts of the emission maximum were observed when C12EOn detergents were utilised. The results correlate with aqueous two-phase partitioning where addition of C12EOn detergents results in more extreme partitioning compared to systems containing only polymers. Dynamic light scattering (DLS) measurements of the EOPO copolymers were carried out, showing that the polymers did not aggregate at concentrations used in aqueous two-phase systems. Quenching of fluorescence with iodide for both proteins and peptide tags was studied. Plots according to the Stern-Volmer equation resulted in a linear fit, indicating exposed tryptophan residues for both free peptides and fusion proteins. The quenching constants were similar for both tagged protein and free peptide tag. The fluorescence results indicated that the tryptophan residues in the tag were exposed to the solvent and could interact with detergents and polymers in the two-phase systems. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Cutinase, Quenching, Protein-peptide fusion, Fluorescence, Aqueous two-phase system, Tryptophan
in
Biochimica et Biophysica Acta - Proteins and Proteomics2002-01-01+01:00
volume
1646
issue
1-2
pages
57 - 66
publisher
Elsevier
external identifiers
  • pmid:12637012
  • wos:000181783400007
  • scopus:1242300064
ISSN
1570-9639
DOI
10.1016/S1570-9639(02)00550-2
language
English
LU publication?
yes
id
cf80fd2e-aadd-430a-83a3-09d4eb531205 (old id 124719)
date added to LUP
2007-07-09 08:19:37
date last changed
2018-05-29 11:03:38
@article{cf80fd2e-aadd-430a-83a3-09d4eb531205,
  abstract     = {Genetic engineering has been used to construct fusion proteins of Fusarium solani pisi cutinase and tryptophan-based tags, expressed in Saccharomyces cerevisiae, to increase the partitioning in aqueous two-phase systems. The separation systems were composed of thermoseparating polymers (random copolymers of ethylene oxide and propylene oxide, EOPO) and detergents (C12EOn). In this study, the fluorescence behaviour of the peptide-tagged protein, free peptide tag and tryptophan was investigated. The tryptophan-tagged proteins, cutinase-(WP)4 and cutinase-TGGSGG-(WP)4, showed emission spectra similar to the free peptides and tryptophan, indicating solvent exposure of the tag. The influence of polymers and detergents on the fluorescence of tagged proteins was examined. When peptides and tagged proteins were exposed to polymer, a slight blue shift of the emission maximum was observed. Larger blue shifts of the emission maximum were observed when C12EOn detergents were utilised. The results correlate with aqueous two-phase partitioning where addition of C12EOn detergents results in more extreme partitioning compared to systems containing only polymers. Dynamic light scattering (DLS) measurements of the EOPO copolymers were carried out, showing that the polymers did not aggregate at concentrations used in aqueous two-phase systems. Quenching of fluorescence with iodide for both proteins and peptide tags was studied. Plots according to the Stern-Volmer equation resulted in a linear fit, indicating exposed tryptophan residues for both free peptides and fusion proteins. The quenching constants were similar for both tagged protein and free peptide tag. The fluorescence results indicated that the tryptophan residues in the tag were exposed to the solvent and could interact with detergents and polymers in the two-phase systems.},
  author       = {Nilsson, Anna and Neves-Petersen, Maria Teresa and Johansson, Hans-Olof and Jansson, Jörgen and Schillén, Karin and Tjerneld, Folke and Petersen, Steffen B},
  issn         = {1570-9639},
  keyword      = {Cutinase,Quenching,Protein-peptide fusion,Fluorescence,Aqueous two-phase system,Tryptophan},
  language     = {eng},
  number       = {1-2},
  pages        = {57--66},
  publisher    = {Elsevier},
  series       = {Biochimica et Biophysica Acta - Proteins and Proteomics2002-01-01+01:00},
  title        = {Tryptophan-tagged cutinase studied by steady state fluorescence for understanding of tag interactions in aqueous two-phase systems},
  url          = {http://dx.doi.org/10.1016/S1570-9639(02)00550-2},
  volume       = {1646},
  year         = {2003},
}