Isopropyl β d thiogalactopyranoside

The E. coli strains carrying the recombinant ChFatB2 and ACP vectors were cultured in Luria-Bertani (LB) broth containing 50 μg/mL ampicillin and Kanamycin, respectively, and induced by 0.5 mM Isopropyl-β-D-thiogalactopyranoside (Takara Biomedical Technology (Beijing) Co., Beijing, China) when the cells had grown to OD₆₀₀ of 0.5 at 37 °C. The cells were further grown at 16 °C for 12 h before being harvested by centrifugation at 6500× g for 20 min. The cell pellet was then suspended in a lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5% glycerol (v/v), and 1 mM β-mercaptoethanol) and homogenized by ultrasonication on ice. The lysate was centrifuged for 30 min at 12,000× g at 4 °C. The supernatant containing soluble target protein was then applied onto Ni-NTA resin (QIAgen, Hilden, Germany). The eluted proteins were further purified on a Superdex 200 column (Cytiva, Marlborough, MA, USA) equilibrated with Tris-HCl buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 5% glycerol (v/v), 1 mM DTT). The fractions containing ChFatB2 were concentrated using an Amicon concentrator 10 kDa (Merck Millipore, Darmstadt, Germany), while ACPs were concentrated using 3 kDa, and the protein concentrations were determined using Bradford assay with BSA as standard.