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Ligand-Directed Lab...
Ligand-Directed Labeling of a Single Lysine Residue in hGST A1-1 Mutants
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- Hederos (Håkansson), Sofia (author)
- Linköpings universitet,Organisk Kemi,Tekniska högskolan
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- Karlsson, Beatrice (author)
- Linköpings universitet,Institutionen för fysik, kemi och biologi,Tekniska högskolan
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Tegler, Lotta (author)
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- Kerstin S., Broo (author)
- Gothenburg University,Göteborgs universitet,Linköpings universitet,Organisk Kemi,Tekniska högskolan,Institutionen för kemi,Department of Chemistry
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(creator_code:org_t)
- 2005-07-02
- 2005
- English.
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In: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 16:4, s. 1009-1018
- Related links:
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http://urn.kb.se/res...
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https://urn.kb.se/re...
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https://doi.org/10.1...
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https://gup.ub.gu.se...
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Abstract
Subject headings
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- Previously, we discovered that human glutathione transferase (hGST) A1-1 could be site-specifically acylated on a tyrosine residue (Y9) to form ester products using thiolesters of glutathione (GS-thiolesters) as acylating reagents. Out of a total of 20 GS-thiolester reagents tested, 15 (75%) are accepted by hGST A1-1 and thus this is a very versatile reaction. The present investigation was aimed at obtaining a more stable product, an amide bond, between the acyl group and the protein, in order to further increase the value of the reaction. Three lysine mutants (Y9K, A216K, and Y9F/A216K) were therefore prepared and screened against a panel of 18 GS-thiolesters. The Y9K mutant did not react with any of the reagents. The double mutant Y9F/A216K reacted with only one reagent, but in contrast, the A216K mutant could be acylated at the introduced lysine 216 with eight (44%) of the GS-thiolesters. The reaction can take place in the presence of glutathione and even in a crude cell lysate for five (28%) of the reagents. Through the screening process we obtained some basic rules relating to reagent requirements. We have thus produced a mutant (A216K) that can be rapidly and site-specifically modified at a lysine residue to form a stable amide linkage with a range of acyl groups. One of the successful reagents is a fluorophore that potentially can be used in downstream protein purification and protein fusion applications.
Subject headings
- NATURVETENSKAP -- Kemi (hsv//swe)
- NATURAL SCIENCES -- Chemical Sciences (hsv//eng)
Keyword
- human GST A1-1
- site-specific covalent modification
- tyrosine 9
- alanine 216
- lysine mutant
- pre-programmed
- ligand-directed protein modification
- intramolecular acyl-transfer reaction
- NATURAL SCIENCES
- NATURVETENSKAP
Publication and Content Type
- ref (subject category)
- art (subject category)
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