Expression and Purification of Recombinant pGEX-4T-1-Linearized-Microcystinase

E. coli BL21 (DE3) cells were transferred with pGEX-4T-1-linearized-microcystinase and plated on LB agar containing 100 μg/ml ampicillin. A single colony was cultured into liquid LB medium with 100 μg/ml ampicillin and shaken at a constant condition of 37°C, 200 rpm. Isopropyl-β-D-thiogalactopyranoside (IPTG), with the final concentration of 0.2 mM, was added to induce protein expression when the optical density at 600 nm was approximately 0.6. Also, the bacterium was constantly incubated at 30°C, 200 rpm for 3 h. The E. coli cells were harvested by centrifuging (8,000 × g, 20 min, 4°C), and the cells were resuspended in phosphate-buffered saline (PBS, pH = 7.0) three times. Cells were placed on ice and disrupted by sonication, and the resultant was centrifuged (15,000 × g, 20 min, 4°C). The supernatant was purified by applying to the GST-Accept nickel column (TaKaRa, Otsu, Japan). The columns were washed twice with the washing buffer at pH 8.0, which contained 50 mM Tris hydrochloride (Tris-HCl) and 150 mM NaCl, and then, the bound proteins were eluted from the columns with an elution buffer (50 mM Tris-HCl, 150 mM NaCl, 250 mM glutathione, pH 8.0). The protein profile was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gel. Also, the concentration of the purified protein was determined at 280 nm using the Ultramicro spectrophotometer (IMPLEN P330, Munich, Germany).