13c d glucose

E.coli codon optimized human COA6 isoform3 was cloned into the pGEX4t-1 vector using BamHI and XhoI restriction endonucleases to generate N-terminal, GST-fused COA6. E.coli codon optimized yeast COA6 was cloned into the pET28a plasmid using XhoI and EcoRI restriction endonucleases to generate N-terminal, His-GFP-fused COA6. The resultant plasmids were then transformed into E.coli BL 21(DE3) cells. Human COA6 and yeast Coa6 expression was induced by the addition of 0.5 mM isopropyl-D-thiogalactopyranoside (IPTG) at an optical density of 600 nm (OD₆₀₀) of 0.6, and the cells were grown at 37°C and 16°C for 4 hr and 12 hr respectively. ¹⁵N/¹³C labeled proteins for NMR structural characterization were expressed in cells grown in minimal medium, which contained 3 g/liter KH₂PO₄, 6.8 g/liter Na₂HPO₄, 0.1 mM CaCl₂, 2 mM MgCl₂, 1x Basal Medium Eagle (BME) vitamin solution (Sigma), 1 g/liter ¹⁵N-ammonium chloride and 4 g/liter ¹³C-D-glucose (Sigma).