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Metal Binding Tags - Characterisation, Use in Bioseparation and Applications of Green Fluorescent Fusion Proteins

Bernaudat, Florent LU (2005)
Abstract
Most of the recombinant proteins produced nowadays are fused to affinity tags, in order to facilitate their purification through affinity chromato-graphy. Out the different tags available, poly-histidine tags are among the most commonly used and can help purification through immobilised metal ion affinity chromatography (IMAC).



In this thesis, we present different metal binding tags, and their affinity towards metals ions was tested using different techniques, such as surface plasmon resonance (SPR), IMAC, immobilised metal affinity gel electrophoresis (IMAGE) or partitioning (IMAP).



SPR was used to compare the binding strength of different poly-histidine tagged lactate dehydrogenases. The technique... (More)
Most of the recombinant proteins produced nowadays are fused to affinity tags, in order to facilitate their purification through affinity chromato-graphy. Out the different tags available, poly-histidine tags are among the most commonly used and can help purification through immobilised metal ion affinity chromatography (IMAC).



In this thesis, we present different metal binding tags, and their affinity towards metals ions was tested using different techniques, such as surface plasmon resonance (SPR), IMAC, immobilised metal affinity gel electrophoresis (IMAGE) or partitioning (IMAP).



SPR was used to compare the binding strength of different poly-histidine tagged lactate dehydrogenases. The technique proved to be fast and effective, and presented also the advantage to only consume small amounts of material.



A peptide library, fused to the green fluorescent protein (GFP), was screened to search for novel metal binding tags, using IMAC in a 96-well plate format and IMAGE. The two methods had their advantages and drawbacks, but in both cases the fusion to GFP was helpful, as it simplified and speeded up the detection of the recombinant proteins.



Affinity tags containing both histidine and hydrophobic residues were found to be very efficient in IMAP, using a PEG/salt aqueous two-phase system. Different amino acid combinations were tested and, for the same number of histidines, the partition coefficient of some of the protein constructs was increased more than twice, reaching K values over 20, when hydrophobic residues were introduced in the tag sequence.



Finally, chimeric fusion proteins combining Fc-binding and fluorescent properties, were evaluated as alternatives to secondary conjugated-antibodies. GFP was fused to one or two repeats of the Z-domain of protein A (Staphylococcus aureus) and expressed in E. coli. His-tagged variants were also prepared, in order to be purified using IMAC, offering a cheap purification method. All constructs were tested in a fluorescent immunoassay test and proved to be as efficient as a commercial fluorescent antibody conjugate. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Kula, Maria-Regina, Germany
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Proteiner, enzymologi, enzymology, E. coli, Immobilised metal ion affinity chromatography, Affinity tags, Green fluorescent protein, Surface plasmon resonance, Genetic engineering, Screening, Proteins, Immunoassay, Biotechnology, Bioteknik
pages
53 pages
publisher
Pure and Applied Biochemistry, Lund University
defense location
Room K:B, Chemical center, Getingevägen 60, Lund Institute of Technology
defense date
2005-06-14 10:15
ISBN
91-628-6450-5
language
English
LU publication?
yes
id
fce4c8f2-3c10-44fc-be68-0edf4049acfe (old id 24509)
date added to LUP
2007-06-05 11:43:59
date last changed
2016-09-19 08:45:13
@phdthesis{fce4c8f2-3c10-44fc-be68-0edf4049acfe,
  abstract     = {Most of the recombinant proteins produced nowadays are fused to affinity tags, in order to facilitate their purification through affinity chromato-graphy. Out the different tags available, poly-histidine tags are among the most commonly used and can help purification through immobilised metal ion affinity chromatography (IMAC).<br/><br>
<br/><br>
In this thesis, we present different metal binding tags, and their affinity towards metals ions was tested using different techniques, such as surface plasmon resonance (SPR), IMAC, immobilised metal affinity gel electrophoresis (IMAGE) or partitioning (IMAP).<br/><br>
<br/><br>
SPR was used to compare the binding strength of different poly-histidine tagged lactate dehydrogenases. The technique proved to be fast and effective, and presented also the advantage to only consume small amounts of material.<br/><br>
<br/><br>
A peptide library, fused to the green fluorescent protein (GFP), was screened to search for novel metal binding tags, using IMAC in a 96-well plate format and IMAGE. The two methods had their advantages and drawbacks, but in both cases the fusion to GFP was helpful, as it simplified and speeded up the detection of the recombinant proteins.<br/><br>
<br/><br>
Affinity tags containing both histidine and hydrophobic residues were found to be very efficient in IMAP, using a PEG/salt aqueous two-phase system. Different amino acid combinations were tested and, for the same number of histidines, the partition coefficient of some of the protein constructs was increased more than twice, reaching K values over 20, when hydrophobic residues were introduced in the tag sequence.<br/><br>
<br/><br>
Finally, chimeric fusion proteins combining Fc-binding and fluorescent properties, were evaluated as alternatives to secondary conjugated-antibodies. GFP was fused to one or two repeats of the Z-domain of protein A (Staphylococcus aureus) and expressed in E. coli. His-tagged variants were also prepared, in order to be purified using IMAC, offering a cheap purification method. All constructs were tested in a fluorescent immunoassay test and proved to be as efficient as a commercial fluorescent antibody conjugate.},
  author       = {Bernaudat, Florent},
  isbn         = {91-628-6450-5},
  keyword      = {Proteiner,enzymologi,enzymology,E. coli,Immobilised metal ion affinity chromatography,Affinity tags,Green fluorescent protein,Surface plasmon resonance,Genetic engineering,Screening,Proteins,Immunoassay,Biotechnology,Bioteknik},
  language     = {eng},
  pages        = {53},
  publisher    = {Pure and Applied Biochemistry, Lund University},
  school       = {Lund University},
  title        = {Metal Binding Tags - Characterisation, Use in Bioseparation and Applications of Green Fluorescent Fusion Proteins},
  year         = {2005},
}