Purification of E. coli holo-ACP and apo-ACP

E. coli holo-ACP and apo-ACP were obtained as previously described⁴⁹. In brief, strain DK754 containing pJT93 (encoding acpS for preparation of holo-ACP) or pJT94 (encoding acpH for preparation of apo-ACP) were inoculated in 10 ml LB medium containing kanamycin, chloramphenicol, and spectinomycin and grown at 37 °C overnight. The starter cultures were transferred into 1 L LB medium containing the same antibiotics and allowed to grow until OD₆₀₀ reached 0.8–1 at 37 °C. Coexpression of ACP with either the AcpS 4’-phosphopantetheinyl transferase or with the AcpH ACP phosphodiesterase were induced by addition of 0.2 mM IPTG followed by incubation at 37 °C for 4 h. Cells were harvested and washed in 10 ml of 50 mM Tris-HCl (pH 8.8). The cells were then resuspended in 10 ml reaction buffer that contained 50 mM Tris-HCl (pH 8.8), 10 mM MgCl₂, 5 mM dithiothreitol, and lysed either by sonication or by three passages through a French pressure cell. The lysates were then cleared by centrifugation at 39,000 × g for 20 min at 4 °C, then incubated at 37 °C for 4 h with or without 1 mM CoA for preparation of holo-ACP or apo-ACP, respectively. Purification of ACP species was performed as previously described⁴⁹.