2.3 Expression of LplA, H protein and the variant HK64A
Cells harboring the plasmids pET28-H, pET28-LplA, and pET28a-HK64A were grown at 37 °C in LB medium containing suitable antibiotics, respectively. For obtaining Hlip, 200 μM lipoic acid were added to the corresponding culture to directly convert the expressed Hapo into Hlip. Induction of the target protein expression was started by adding 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) when the optical density of the culture at 600 nm reached 0.6. Then, the culture was incubated for an additional 12 h at 30 °C. After the medium removal by centrifugation (10,000 x g, 5 minutes, 4 °C), the bacterial pellet was re-suspended in Tris-HCl buffer (50 mM, pH 7.5) and lysed using a Xinzhi JY92-IIN Ultrasonic Homogenizer. The supernatant (lysate) was collected by centrifugation at 10,000 x g for 5 min at 4 °C and stored at 4 °C for the following protein purification step. Protein expression was examined using SDS-PAGE.