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.