liu.seSearch for publications in DiVA
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • oxford
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Surface-Assisted Delivery of Fluorescent Groups to hGST A1-1 and a Lysine Mutant
Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry . Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry . Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry . Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry . Linköping University, The Institute of Technology.
2006 (English)In: Bioconjugate Chemistry, ISSN 1043-1802, Vol. 17, no 2, 429-437 p.Article in journal (Refereed) Published
Abstract [en]

Human glutathione transferase (hGST) A1-1 and a lysine mutant (A216K) can both be rapidly and site-specifically acylated on Y9 and K216, respectively, using a range of thiolesters of glutathione (GS-thiolesters) as modifying reagents. The present investigation was aimed at developing a method with which to deliver a fluorescent acyl group from a solid support under conditions compatible with standard protein purification schemes. A number of fluorescent GS-thiolesters with modified peptide backbones were therefore prepared and tested for reactivity toward hGST A1-1 and the A216K mutant. Substitutions at the α-NH2 part of the glutathione backbone were not tolerated by the proteins. However, two fluorescent reagents that carry a biotin moiety at the C-terminal part of glutathione were found through MALDI-MS experiments to react in solution with Y9 of the wild-type protein and one reagent with K216 of A216K. The reaction can take place in the presence of glutathione and even in a crude E. coli lysate of cells expressing A216K. Delivery of the fluorescent group to Y9 or K216 was possible using NeutrAvidin (NA) beads that had been preincubated with biotinylated reagent. Alternatively, excess reagent can be removed by a brief incubation with NA beads. We have thus now developed a system for protein labeling with easy removal of excess and used up low-molecular weight reagent. This strategy can conceivably be utilized in future protein purification and labeling experiments.

Place, publisher, year, edition, pages
2006. Vol. 17, no 2, 429-437 p.
Keyword [en]
human GST A1-1, site-specific covalent modification, tyrosine 9, alanine 216, lysine 216, pre-programmed, solid support delivery, biotin, streptavidin
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-13197DOI: 10.1021/bc0502762OAI: oai:DiVA.org:liu-13197DiVA: diva2:18021
Available from: 2008-04-28 Created: 2008-04-28 Last updated: 2009-06-08
In thesis
1. A Novel Route for Construction of Multipurpose Receptors through Chemical Modification of Glutathione Transferases
Open this publication in new window or tab >>A Novel Route for Construction of Multipurpose Receptors through Chemical Modification of Glutathione Transferases
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes how the human Alpha class glutathione transferase (GST) A1-1 can be reprogrammed either to function as a multipurpose biosensor for detection of small molecule analytes, or as a handle providing for more efficient protein purification.

A novel, user-friendly, and efficient method for site-specific introduction of functional groups into the active site of hGST A1-1 is the platform for these achievements. The designed thioester reagents are glutathione-based and they are able to label one single nucleophile (Y9) and leave the other 50 nucleophiles (in hGST A1-1) intact. The modification reaction was tested with five classes of GSTs (Alpha, Mu, Pi, Theta and Omega) and was found to be specific for the Alpha class isoenzymes. The reaction was further refined to target a single lysine residue, K216 in the hGST A1-1 mutant A216K, providing a stable amide bond between the protein and the labeling group. To further improve the labeling process, biotinylated reagents that could deliver the acyl group to Y9 (wt hGST A1-1) or K216 in the lysine mutant, while attached to streptavidin-coated agarose beads, were designed and synthesized.

A focused library of eleven A216K/M208X mutants was made via random mutagenesis to provide an array of proteins with altered micro-environments in the hydrophobic binding site, where M208 is situated. Through the invented route for site-specific labeling, a fluorescent probe (coumarin) was introduced on K216 in all double mutants, with the purpose of developing a protein-based biosensor, akin to the olfactory system. The array of coumarin-labeled proteins responded differently to the addition of different analytes, and the responses were analyzed through pattern recognition of the fluorescence signals. The labeled proteins could also be site-specifically immobilized on a PEG-based biosensor chip via the single C112 on the surface of the protein, enabling development of surface-based biosensing systems.

Also, a refined system for efficient detection and purification of GST-fusion proteins is presented. Through a screening process involving A216K and all produced A216K/M208X mutants, two candidates (A216K and A216K/M208F) were singled out as scaffolds for the next generation of fusion proteins. In addition to the features present in commercially available GST fusion constructs, the new mutants can be site-specifically labeled with a fluorophore in bacterial lysates providing quick and sensitive monitoring of expression and purification. Furthermore, the proteins could be labeled with a unique aldehyde moiety providing for a novel protein purification scheme.

Place, publisher, year, edition, pages
Institutionen för teknik och naturvetenskap, 2008. 79 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1184
Keyword
Human GST A1-1, site-specific covalent modification, tyrosine 9, lysine 216, methionine 208, multipurpose receptor, pattern recognition, protein purification
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-11612 (URN)978-91-7393-893-8 (ISBN)
Public defence
2008-05-23, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 10:15 (English)
Opponent
Supervisors
Available from: 2008-04-28 Created: 2008-04-28 Last updated: 2009-05-18

Open Access in DiVA

No full text

Other links

Publisher's full textLink to Ph.D. thesis

Authority records BETA

Viljanen, JohanBroo, Kerstin S.

Search in DiVA

By author/editor
Viljanen, JohanBroo, Kerstin S.
By organisation
Organic Chemistry The Institute of Technology
Natural Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 77 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • oxford
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf