Employment of colorimetric enzyme assay for monitoring expression and solubility of GST fusion proteins targeted to inclusion bodies

High levels of recombinant protein expression can lead to the formation of insoluble inclusion bodies. These complex aggregates are commonly solubilized in strong denaturants, such as 6-8 M urea, although, if possible, solubilization under milder conditions could facilitate subsequent refolding and purification of bioactive proteins. Commercially available GST-tag assays are designed for quantitative measurement of GST activity under native conditions. GST fusion proteins accumulated in inclusion bodies are considered to be undetectable by such assays. In this work, solubilization of recombinantly produced proteins was performed in 4 M urea. The activity of rGST was assayed in 2 M urea and it was shown that rGST preserves 85% of its activity under such denaturing conditions. A colorimetric GST activity assay with 1-chloro-2, 4-dinitrobenzene (CDNB) was examined for use in rapid detection of expression targeted to inclusion bodies and for the identification of inclusion body proteins which can be solubilized in low concentrations of chaotropic agents. Applicability of the assay was evaluated by tracking protein expression of two GST-fused allergens of biopharmaceutical value in E. coli, GST-Der p 2 and GST-Mus a 5, both targeted to inclusion bodies

Detection of protein S-sulfhydration by a tag-switch technique

Protein S-sulfhydration (forming -S-SH adducts from cysteine residues) is a newly defined oxidative posttranslational modification and plays an important role in H2 S-mediated signaling pathways. In this study we report the first selective, "tag-switch" method which can directly label protein S-sulfhydrated residues by forming stable thioether conjugates. Furthermore we demonstrate that H2 S alone cannot lead to S-sulfhydration and that the two possible physiological mechanisms include reaction with protein sulfenic acids (P-SOH) or the involvement of metal centers which would facilitate the oxidation of H2 S to HS(.)