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

Direct link
Orthogonal Protein Purification - Expanding the Repertoire of GST Fusion Systems
Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry . Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
Göteborg University.
2008 (English)In: Protein Expression & Purification, ISSN 1046-5928, Vol. 57, no 1, 17-26 p.Article in journal (Refereed) Published
Abstract [en]

We have previously developed a labeling scheme that can be used to site-specifically link human glutathione transferases (hGSTs) from the alpha class to chemical entities such as fluorophores and aldehydes. The reagents are in-house synthesized derivatives of glutathione (GS-derivatives). We have focused on a lysine mutant of hGST A1:A216K. In this study, we wanted to utilize these findings and improve on protein purification schemes that are using GSTs as fusion partners. We have used random mutagenesis to scramble the hydrophobic binding site of A216K through mutations at position M208 and isolated a library of 11 A216K/M208X mutants. All mutants were easily expressed and purified and retained all or parts of the catalytic properties of the parent GST. The mutants were stable over several days at room temperature. The A216K/M208X mutants could be site-specifically labeled using our designed fluorescent reagents. Furthermore, reaction with an aldehyde-containing reagent termed GS-Al results in site-specific introduction of an orthogonal handle for subsequent conjugation with aldehyde-reactive probes. Labeling with coumarin results in a fluorescent protein-conjugate that can bind glutathione (GSH) derivatives for subsequent affinity purification. The Kd for S-hexyl-GSH of coumarin-labeled A216K was measured to be 2.5 μM. The candidate proteins A216K and A216K/M208F could be purified in high yield in a one-step procedure through affinity chromatography (Glutathione Sepharose™ 4B). The proteins can readily be perceived as improved GST fusion partners.

Place, publisher, year, edition, pages
2008. Vol. 57, no 1, 17-26 p.
Keyword [en]
Human GST A1-1 mutants; Site-specific covalent modification; Lysine 216; Methionine 208; Protein purification; Fusion partner
National Category
Natural Sciences
URN: urn:nbn:se:liu:diva-13199DOI: 10.1016/j.pep.2007.09.011OAI: diva2:18023
Available from: 2008-04-28 Created: 2008-04-28 Last updated: 2009-05-18
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.
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1184
Human GST A1-1, site-specific covalent modification, tyrosine 9, lysine 216, methionine 208, multipurpose receptor, pattern recognition, protein purification
National Category
Chemical Sciences
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)
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

Search in DiVA

By author/editor
Viljanen, Johan
By organisation
Organic Chemistry The Institute of TechnologyDepartment of Physics, Chemistry and Biology
Natural Sciences

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 92 hits
ReferencesLink to record
Permanent link

Direct link