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Hydrophobic Interactions in Bioseparation - Applications of Modified Green Fluorescent Protein

Becker, Kristian LU (2008)
Abstract
This thesis describes interactions that are salt-promoted and mainly based on the hydrophobicity of proteins. A series of variants of the green fluorescent protein (GFP) was first constructed by site-directed mutagenesis. Several tags with various properties were then genetically fused to the N-terminus of GFP. The protein variants constructed had small characteristic differences in hydrophobicity, charge and metal affinity. These variants were then used to develop separation media and methods, and for the characterization of the proteins themselves for various applications.



A mild pH-responsive hydrophobic medium was characterized, which was able to resolve the protein variants, down to just one difference in the amino... (More)
This thesis describes interactions that are salt-promoted and mainly based on the hydrophobicity of proteins. A series of variants of the green fluorescent protein (GFP) was first constructed by site-directed mutagenesis. Several tags with various properties were then genetically fused to the N-terminus of GFP. The protein variants constructed had small characteristic differences in hydrophobicity, charge and metal affinity. These variants were then used to develop separation media and methods, and for the characterization of the proteins themselves for various applications.



A mild pH-responsive hydrophobic medium was characterized, which was able to resolve the protein variants, down to just one difference in the amino acid sequence in hydrophobic interaction chromatography. The results were compared with theoretical calculations of hydrophobicity, and found to correlate well. Furthermore, a multifunctional purification tag was developed for use in protein isolation using various techniques. The tag, consisting of hydrophobic, charged, metal-binding amino acid residues, bound its protein host more strongly than the corresponding non-tagged protein when using a number of relevant isolation techniques.



The isolation technique was scaled down to capillaries where variants differing in charge and hydrophobicity could be resolved using nanoparticles as the stationary phase in capillary electrochromatography.



Finally, some of the GFP variants were tested regarding their metal affinity and the quenching of fluorescence by metal ions in the development of a biosensor. It was found that the accessibility of histidine residues on the surface of the protein is important for the quenching effect. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Prof Queiroz, Joao, Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Hydrophobic interaction chromatography, capillary electrochromatography, E. coli, isolation tags, haemoglobin, green fluorescent protein, retention prediction, genetic engineering, accessible surface area
pages
123 pages
defense location
Lecture hall B, Centre for chemistry and chemical engineering, Getingevägen 60, Lund University Faculty of Engineering
defense date
2008-11-21 10:15
ISBN
978-91-628-7625-8
language
English
LU publication?
yes
id
16591b86-2872-4c98-be59-1292b180240b (old id 1258790)
date added to LUP
2008-10-27 11:18:55
date last changed
2016-09-19 08:45:18
@misc{16591b86-2872-4c98-be59-1292b180240b,
  abstract     = {This thesis describes interactions that are salt-promoted and mainly based on the hydrophobicity of proteins. A series of variants of the green fluorescent protein (GFP) was first constructed by site-directed mutagenesis. Several tags with various properties were then genetically fused to the N-terminus of GFP. The protein variants constructed had small characteristic differences in hydrophobicity, charge and metal affinity. These variants were then used to develop separation media and methods, and for the characterization of the proteins themselves for various applications.<br/><br>
<br/><br>
A mild pH-responsive hydrophobic medium was characterized, which was able to resolve the protein variants, down to just one difference in the amino acid sequence in hydrophobic interaction chromatography. The results were compared with theoretical calculations of hydrophobicity, and found to correlate well. Furthermore, a multifunctional purification tag was developed for use in protein isolation using various techniques. The tag, consisting of hydrophobic, charged, metal-binding amino acid residues, bound its protein host more strongly than the corresponding non-tagged protein when using a number of relevant isolation techniques.<br/><br>
<br/><br>
The isolation technique was scaled down to capillaries where variants differing in charge and hydrophobicity could be resolved using nanoparticles as the stationary phase in capillary electrochromatography.<br/><br>
<br/><br>
Finally, some of the GFP variants were tested regarding their metal affinity and the quenching of fluorescence by metal ions in the development of a biosensor. It was found that the accessibility of histidine residues on the surface of the protein is important for the quenching effect.},
  author       = {Becker, Kristian},
  isbn         = {978-91-628-7625-8},
  keyword      = {Hydrophobic interaction chromatography,capillary electrochromatography,E. coli,isolation tags,haemoglobin,green fluorescent protein,retention prediction,genetic engineering,accessible surface area},
  language     = {eng},
  pages        = {123},
  title        = {Hydrophobic Interactions in Bioseparation - Applications of Modified Green Fluorescent Protein},
  year         = {2008},
}