Ligand-Directed Labeling of a Single Lysine Residue in hGST A1-1 Mutants
2005 (English)In: Bioconjugate chemistry, ISSN 1043-1802 (print) 1520-4812 (online), Vol. 16, no 4, 1009-1018 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2005. Vol. 16, no 4, 1009-1018 p.
human GST A1-1, site-specific covalent modification, tyrosine 9, alanine 216, lysine mutant, pre-programmed, ligand-directed protein modification, intramolecular acyl-transfer reaction
IdentifiersURN: urn:nbn:se:liu:diva-13367DOI: 10.1021/bc050111tOAI: oai:DiVA.org:liu-13367DiVA: diva2:20490