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Cutinase-peptide fusions in thermoseparating aqueous two-phase systems - Prediction of partitioning and enhanced tag efficiency by detergent addition

Nilsson, Anna LU ; Mannesse, Maurice; Egmond, Maarten R and Tjerneld, Folke LU (2002) In Journal of Chromatography A 946(1-2). p.141-155
Abstract
It is of increasing importance to develop efficient purification methods for recombinant proteins where the number of steps can be minimised. The aim has been to establish a method for predicting the partitioning of the wild-type target protein in an aqueous two-phase system, and with this as basis, develop fusion tags and optimise the phase system for enhanced partitioning of the target protein. The surface of the lipolytic enzyme cutinase from Fusarium solani pisi was investigated with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP). The accessible surface areas for the different amino acid residues were used together with peptide partitioning data to calculate the partition coefficient for the... (More)
It is of increasing importance to develop efficient purification methods for recombinant proteins where the number of steps can be minimised. The aim has been to establish a method for predicting the partitioning of the wild-type target protein in an aqueous two-phase system, and with this as basis, develop fusion tags and optimise the phase system for enhanced partitioning of the target protein. The surface of the lipolytic enzyme cutinase from Fusarium solani pisi was investigated with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP). The accessible surface areas for the different amino acid residues were used together with peptide partitioning data to calculate the partition coefficient for the protein. The separation system was composed of a thermoseparating random copolymer of ethylene oxide and propylene oxide, Breox PAG 50A 1000, as top phase forming polymer and a hydroxypropyl starch polymer, Reppal PES 200, as bottom phase polymer. The calculated partition coefficient for the wild-type protein (K=1.0) agreed reasonably well with the experimentally determined value (K=0.85). Genetic engineering was used to construct fusion proteins expressed in Saccharomyces cerevisiae based on cutinase and peptide tags containing tryptophan, to enhance the partitioning in aqueous two-phase systems. The partitioning of the cutinase constructs could qualitatively be predicted from peptide partitioning data, i.e. the trends in partitioning could be predicted. A spacer peptide introduced between protein and tag increased the partitioning of the protein towards the ethylene oxide-propylene oxide (EOPO) copolymer top phase. The aqueous two-phase system was modified by addition of detergent to increase the partitioning of the cutinase variants towards the EOPO copolymer phase. Triton and a series of C12En detergents selectively increased the partitioning of cutinase constructs with (WP)4-based tags up to 14 times compared to wild-type cutinase. The protein partition could almost quantitatively be predicted from the peptide partition data. (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
Peptides, Cutinase, Trytophan, Polymers, Proteins
in
Journal of Chromatography A
volume
946
issue
1-2
pages
141 - 155
publisher
Elsevier
external identifiers
  • wos:000173820700014
  • pmid:11873963
  • scopus:0037039743
ISSN
0021-9673
DOI
10.1016/S0021-9673(01)01543-6
language
English
LU publication?
yes
id
f75a5852-01e5-4d36-8839-18f353c227f9 (old id 124862)
date added to LUP
2007-07-06 11:52:49
date last changed
2017-01-01 06:52:18
@article{f75a5852-01e5-4d36-8839-18f353c227f9,
  abstract     = {It is of increasing importance to develop efficient purification methods for recombinant proteins where the number of steps can be minimised. The aim has been to establish a method for predicting the partitioning of the wild-type target protein in an aqueous two-phase system, and with this as basis, develop fusion tags and optimise the phase system for enhanced partitioning of the target protein. The surface of the lipolytic enzyme cutinase from Fusarium solani pisi was investigated with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP). The accessible surface areas for the different amino acid residues were used together with peptide partitioning data to calculate the partition coefficient for the protein. The separation system was composed of a thermoseparating random copolymer of ethylene oxide and propylene oxide, Breox PAG 50A 1000, as top phase forming polymer and a hydroxypropyl starch polymer, Reppal PES 200, as bottom phase polymer. The calculated partition coefficient for the wild-type protein (K=1.0) agreed reasonably well with the experimentally determined value (K=0.85). Genetic engineering was used to construct fusion proteins expressed in Saccharomyces cerevisiae based on cutinase and peptide tags containing tryptophan, to enhance the partitioning in aqueous two-phase systems. The partitioning of the cutinase constructs could qualitatively be predicted from peptide partitioning data, i.e. the trends in partitioning could be predicted. A spacer peptide introduced between protein and tag increased the partitioning of the protein towards the ethylene oxide-propylene oxide (EOPO) copolymer top phase. The aqueous two-phase system was modified by addition of detergent to increase the partitioning of the cutinase variants towards the EOPO copolymer phase. Triton and a series of C12En detergents selectively increased the partitioning of cutinase constructs with (WP)4-based tags up to 14 times compared to wild-type cutinase. The protein partition could almost quantitatively be predicted from the peptide partition data.},
  author       = {Nilsson, Anna and Mannesse, Maurice and Egmond, Maarten R and Tjerneld, Folke},
  issn         = {0021-9673},
  keyword      = {Peptides,Cutinase,Trytophan,Polymers,Proteins},
  language     = {eng},
  number       = {1-2},
  pages        = {141--155},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography A},
  title        = {Cutinase-peptide fusions in thermoseparating aqueous two-phase systems - Prediction of partitioning and enhanced tag efficiency by detergent addition},
  url          = {http://dx.doi.org/10.1016/S0021-9673(01)01543-6},
  volume       = {946},
  year         = {2002},
}