Ligand-Directed Labeling of a Single Lysine Residue in hGST A1-1 Mutants

Hederos (Håkansson), SofiaLinköpings universitet,Organisk Kemi,Tekniska högskolan (författare)

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.

Karlsson, BeatriceLinköpings universitet,Institutionen för fysik, kemi och biologi,Tekniska högskolan(Swepub:liu)beaka29 (författare)
Tegler, Lotta (författare)
Kerstin S., BrooGothenburg University,Göteborgs universitet,Linköpings universitet,Organisk Kemi,Tekniska högskolan,Institutionen för kemi,Department of Chemistry(Swepub:gu)xbroke (författare)
Linköpings universitetOrganisk Kemi (creator_code:org_t)

Ingår i:Bioconjugate chemistry: American Chemical Society (ACS)16:4, s. 1009-10181043-18021520-4812

Av författaren/redakt...: Hederos (Håkanss ...; Karlsson, Beatri ...; Tegler, Lotta; Kerstin S., Broo

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