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

详细信息    查看全文

• 作者：Sofia Hederos ; Beatrice Karlsson ; Lotta Tegler ; and Kerstin S. Broo
• 刊名：Bioconjugate Chemistry
• 出版年：2005
• 出版时间：July 2005
• 年：2005
• 卷：16
• 期：4
• 页码：1009 - 1018
• 全文大小：569K
• 年卷期：v.16,no.4(July 2005)
• ISSN：1520-4812

文摘

Previously, we discovered that human glutathione transferase (hGST) A1-1 could be site-specificallyacylated 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%) areaccepted by hGST A1-1 and thus this is a very versatile reaction. The present investigation was aimedat obtaining a more stable product, an amide bond, between the acyl group and the protein, in orderto 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 notreact with any of the reagents. The double mutant Y9F/A216K reacted with only one reagent, but incontrast, the A216K mutant could be acylated at the introduced lysine 216 with eight (44%) of theGS-thiolesters. The reaction can take place in the presence of glutathione and even in a crude celllysate for five (28%) of the reagents. Through the screening process we obtained some basic rulesrelating to reagent requirements. We have thus produced a mutant (A216K) that can be rapidly andsite-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 proteinpurification and protein fusion applications.