*4.5. Protein Purification by Metal Chelate A*ffi*nity Chromatography*

After harvest and cell lysis, heterologously expressed 5xHis-PK proteins were purified by Ni-NTA affinity chromatography in a batch procedure. The cleared lysate was mixed for 1 h at 4 ◦C together with 2 mL Ni-NTA agarose beads (Macherey Nagel, Düren, Germany), which had been washed earlier with lysis buffer (50 mM TRIS-HCl pH 8, 300 mM NaCl, 1 mM imidazole). The supernatant obtained by centrifugation at 300× *g* for 5 minutes at 4 ◦C was removed. Afterwards, the Ni-NTA beads were resuspended in 4 mL wash buffer (50 mM TRIS-HCl pH 8, 300 mM NaCl, 2 mM imidazole) and added to a self-made column plugged with cotton wool. The flow through was collected and a further washing step followed. Matrix-bound proteins were eluted stepwise by application of four times

0.5 mL elution buffer (50 mM TRIS-HCl pH 8, 300 mM NaCl, 250 mM imidazole). The obtained elution fractions were desalted afterwards.
