E. coli BL21 (DE3)pLysS cells (Invitrogen, Carlsbad, CA, USA) were transformed with plasmid DNA. Overnight cultures were used to inoculate 2 L lysogeny broth medium supplemented with 50 µg/mL kanamycin and 20 µg/mL chloramphenicol. Protein expression was induced in the exponential phase by the addition of isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.4 mM. Induced cells were grown at 37 °C and 140 rpm for 4 h. Cells were harvested for 15 min at 5000× g and resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl and 10 mM imidazole, pH 8.0). Cells were disrupted using lysozyme digestion followed by sonication (HF-Generator GM2070 in combination with an ultrasonic converter UW2070, standard horn SH 70G, and microtip MS73, Bandelin, Berlin, Germany). The soluble and insoluble fractions were separated by centrifugation at 20,000× g for 45 min. His-tagged proteins were incubated with nickel-nitrilotriacetic acid (Ni-NTA) sepharose (Qiagen, Hilden, Germany) at 4 °C with slight shaking for 1 h. Washing was performed with wash buffer I (50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole, pH 8.0) and subsequently with wash buffer 2 (50 mM NaH2PO4, 300 mM NaCl, 20 (v/v) glycerol, and 50 mM imidazole, pH 8.0) until the OD280 reached zero. Elution was achieved with elution buffer (50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole, pH 8.0). Protein containing fractions were pooled and dialyzed against 40 mM K-Pi, pH 7.2. After dialysis, protein concentrations were determined using a molar extinction coefficient of 8480 M−1 cm−1 for PRX-IIE and its variants and 9970 M−1·cm−1 and 4470 M−1·cm−1 for GRX-S12 and SRX, respectively. GRX-C5 (At4g28730) was purified following established procedures [18,19].

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