Overexpression and Purification of KDPG Aldolase of Synechocystis sp. PCC 6803.

For homologous overexpression of the KDPG aldolase (sll0107), ORF was cloned into p2M2-Pcpc560ter (14) with a C-terminal His₆-tag (for primers, see Table S3) and transformed into Δeda, resulting in the mutant Δeda/cpc::eda^{Synechocystis}. To purify the enzyme cells of the overexpression strain, Δeda/cpc::eda^{Synechocystis} were harvested, resuspended in 100 mM Tris at pH 8.0 and 150 mM NaCl, and broken with glass beads (diameter, 0.17–0.18 mm) by vortexing 10 min at 4 °C. After centrifugation at 800 × g for 1 min at 4 °C, the liquid phase was removed and centrifuged at 1,300 × g for 10 min at 4 °C. The resulting homogenate was checked for unbroken cells under the microscope and, if necessary, centrifuged a second time at 1,300 × g for 10 min at 4 °C to remove unbroken cells. The homogenate was applied on a Ni-NTA column. After it completely entered the column, it was washed four times with two column volumes (cvs) wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole at pH 8). Subsequently, the protein was eluted from the column by applying six times 0.5 cvs elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole at pH 8) and collecting the eluate in 0.5-cv fractions.