Fig. 1. Schematic showing the two methods used to produce acyl-ACP. (A) Expression of the acpP gene gives rise to apo-ACP. (B) Conversion of apo-ACP to holo-ACP by action of holo-ACP synthase. (C) In vitro acylation with free fatty acid by reaction with acyl-ACP synthetase [14]. (D) Direct in vitro conversion of apo-ACP to acyl-ACP by the reaction of holo-ACP synthase in the presence of acyl-CoA [15].

Fig. 2. (A) Ampicillin-selectable pSACP-2a plasmid originally created for expression of spinach ACP [26]. (B) Tetracycline-selectable pSACP-2t plasmid found to be suitable for expression of spinach ACP in a chemically defined medium required for isotopic labeling in preparation for NMR structural studies. The positions of restriction sites used for cloning and digestion mapping are indicated.

Fig. 3. Time course of the fermentation used to express spinach ACP. The solid line shows the agitation rate while the dashed line shows the dissolved O₂. The arrow at 10 h shows the point at which the initially added glucose was judged to be depleted and at which re-feeding, induction of protein expression, and temperature change were initiated. The arrow at 15.5 h shows where the cells were harvested.

Fig. 4. Time course of the expression of spinach ACP as monitored by denaturing gel electrophoresis. Lane 1, corresponds to molecular weight markers; lane 2, is an analysis of cellular proteins present at the time of induction (10 h in Fig. 3); lanes 3–5 are analyses of cellular proteins obtained from cells harvested at, respectively, at 1, 3, and 5 h after induction. The amount of protein loaded in each lane was normalized to the cell density indicated by OD₆₀₀ measurements. The arrow indicates the position of the ACP on the gel.

Fig. 5. Purification of spinach ACP as monitored by denaturing gel electrophoresis. Lane 1, corresponds to molecular weight markers; lane 2, is an analysis of the cell lysate; lane 3, contained the re-suspended pellet from the 50% PEG 3350 precipitation; lane 4, contained purified apo-ACP obtained from anion exchange chromatography. The arrow indicates the position of ACP on the gel.

Fig. 6. Five hundred megahertz 2D ¹H–¹⁵N HSQC spectra of 2 mM spinach 10:0-ACP (blue) and 18:0-ACP (red) with >98% enrichment of ¹⁵ N in the protein (the phosphopantetheine group was not ¹⁵N-labeled in these samples). The sample temperature was controlled at 14 °C. The solution conditions were 10 mM Mes buffer, pH 6.1, containing 100 mM NaCl. The ¹H and ¹⁵N carrier were positioned at 4.92 ppm (position of the water signal) and 118.2 ppm, respectively. A total of eight transients of 128(t₁) × 1024(t₂) complex data points were collected with spectral widths of 13 ppm for ¹H and 33 ppm for ¹⁵N.

Purification of the apo form of spinach acyl carrier protein