Purification and Characterization of D-Lac Variant Protein Expressed in E. coli BL21(DE3)

The D-Lac variant plasmid was introduced into E. coli BL21(DE3) cells, which were cultured according to established protocols. The cells were then harvested by refrigerated centrifugation at 5000 g for 10 minutes and stored at -20°C. Ultrasonic lysis was employed using a state-of-the-art ultrasound processor. The resulting cell lysate was purified by Nickel-affinity chromatography in an AKTA avant (GE Healthcare, USA) using buffer A (25 mM Tris, 100 mM NaCl, 20 mM imidazole, pH 7.5) and buffer B (25 mM Tris, 100 mM NaCl, 20 mM imidazole, 7.5). The protein concentration was determined using the Bradford Coomassie brilliant blue method, which involved measuring the absorbance at 590 nm with a microplate reader.