Isopropyl thio β d galactoside iptg

Escherichia coli BL21(DE3) (F^–ompT hsdSB (r_B^–m_B^–) gal dcm) (Shanghai Weidi Biotechnology Co., Ltd., Shanghai, China) and E. coli DH5α (dlacZ Delta M15 Delta (lacZYA-argF) U169 recA1 endA1 hsdR17(rK-mK+) supE44 thi-1 gyrA96 relA1) were used for protein expression and cloning, respectively (Studier and Moffatt, 1986 (link); Taylor et al., 1993 (link)). A prepacked desalting column and Ni Sepharose column were purchased from Smart Lifesciences (Changzhou China). The pET28a+ vector from Personalbio (Shanghai, China) was used to express recombinant proteins. Isopropylthio-β-D-galactoside (IPTG) and kanamycin were obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). Ethylenediaminetetraacetic acid (EDTA), 1,4-dithiothreitol (DTT), 5,5′dithiobis-(2-nitrobenzoic acid) (DTNB), acetyl coenzyme A trilithium salt (AcCoA), and enzyme substrates including 2-aminofluorene (2-AF), hydralazine (HDZ), 5-aminosalicylate (5-AS), 4-amino salicylic (4-AS), sulfamethoxazole (SMX), INH, 4-chloro-3-methylaniline (4-C3ME) and 4-aminobenzoic acid (pABA) were purchased from Aladdin (Shanghai, China).

The DNA fragments encoding full-length CtrA and Tr1 were cloned into pET-33b(+) for recombinant CtrA (rCtrA) and recombinant Tr1 (rTr1), pET-41a(+) for GST-rCtrA as described previously (Cheng et al., 2006 (link)). Escherichia coli BL21 (DE3) cells were transformed with plasmids to express the recombinant proteins. The strains were induced to express rCtrA with 1 mM isopropyl-thio-β-D-galactoside (IPTG; Solarbio, Beijing, China) at 37°C for 4 h, or to express rTr1 with 1 mM IPTG at 20°C for 5 h, or to express GST and GST-rCtrA with 0.1 mM IPTG at 37°C for 4 h. rCtrA and rTr1 were purified from E. coli inclusion bodies using 6 M urea, and GST and GST-rCtrA were purified from E. coli soluble fraction (Cheng et al., 2006 (link)). The purified proteins were dialyzed against stocking buffer [10 mM Tris-HCl (Genview, Beijing, China), pH 7.5, 1 mM dithiothreitol (DTT; Solarbio)] for further experiments.

The DNA fragment encoding full-length CtrA was cloned into pET-41a(+) to express recombinant CtrA (GST-rCtrA) as described previously (Cheng et al., 2006 (link)). The DNA fragments encoding GshA, GshB and GST were amplified using specific primers (Supplementary Table S1). The gshA fragment was cloned into pET-His-SUMO to express recombinant GshA (SUMO-rGshA) with an N-terminal His-tag. The fragment of gshB or gst was cloned into pET-33b(+) to express recombinant GshB (rGshB) or GST (rGST) with an N-terminal His-tag, respectively. The constructed plasmids were transformed into E. coli DH5α cells, extracted and confirmed by sequencing. E. coli BL21 (DE3) cells were transformed with the resulting plasmids to express SUMO-rGshA, rGshB and rGST respectively, with pET-41a(+) to express GST or pET-His-SUMO to express SUMO. E. coli BL21 (DE3) cells were induced to express recombinant proteins at an OD₆₀₀ of 0.6 with 0.1 mM isopropyl-thio-β-D-galactoside (IPTG; Solarbio, Beijing, China) at 37°C for 4 h. All proteins were purified from soluble fraction with Ni-affinity chromatography and concentrated using an Amicon Ultra-0.5 Centrifugal Filter Unit (10 kDa MWCO).