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 Al: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 out-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 K-d for S-hexyl-GSH of coumarin-labeled A216K was measured to be 2.5 mu M. The candidate proteins A216K and A216K/M208F could be purified in high yield in a one-step procedure through affinity chromatography (Glutathione Sepharose (TM) 4B). The proteins can readily be perceived as improved GST fusion partners. (c) 2007 Elsevier Inc. All rights reserved.