Glutathione sepharose 4 fast flow column

The sequence coding for 6×His tag-labelled ΔMKD1 (ΔMKD1) was amplified by RT-PCR using specific primers (see Supplementary Table S1). The amplified PCR products were introduced into SgfI and PmeI sites of the pF3KWG (BYDV) Flexi plasmid (Promega). The ΔMKD1 protein was synthesized using the TNT SP6 High-Yield Wheat Germ Protein Expression System (Promega). In vitro transcription/translation was performed according to the manufacturer’s instructions. The ΔMKD1 protein was purified using a Ni Sepharose High-Performance column (GE Healthcare). MKK1, MKK2, and MKK5 were amplified from cDNA by PCR using specific primers (Supplementary Table S1). Amplified fragments of MKK1, MKK2, and MKK5 were cloned into SmaI and NotI (blunt ended) sites of the pGEX6p-1 plasmid (GE Healthcare). MKK1, MKK2, and MKK5 plasmids were transformed into E. coli BL21-CodonPlus (DE3)-RIL (Agilent Technologies). The recombinant proteins glutathione S-transferase (GST)–MKK1, GST–MKK2, and GST–MKK5 were digested by PreScission Protease (GE Healthcare). The resulting MKK1, MKK2, and MKK5 proteins were purified using a glutathione Sepharose 4 Fast Flow column (GE Healthcare).

GST activity was measured according to the method described by Habdous et al. [29 (link)] with some modifications. The His-AfGST (0.5 μg) in 30 μL of 0.1 M phosphate buffer (PB) (pH 6.5) was mixed with cyclo(l-Ala-l-Pro) or ethacrynic acid and incubated at 4 °C for 1 h. After incubation, 470 μL 0.1 M PB (pH 6.5) containing CDNB and GSH was added to the mixture. The final concentration of CDNB, GSH, cyclo(l-Ala-l-Pro), and ethacrynic acid were 1 mM, 1 mM, 2 mM, and 100 μM, respectively. Enzyme activity was calculated according to the change in an absorbance at 340 nm after 5 min. A control experiment was performed without cyclo(l-Ala-l-Pro) or ethacrynic acid.
The vector used to express GST-tagged fusion protein (pGEX-6P-1) was used to prepare rShGST. E. coli BL21 (DE3) cells containing pGEX-6P-1 were cultured as described in Section 5.5, and rShGST was purified with a Glutathione Sepharose 4 Fast Flow column (GE Healthcare, Little Chalfont, UK) according to the manufacturer’s protocol. rShGST was stored as 20% glycerol solution at −80 °C.