2.2. Optimization of purification process

DX2 expressed in Escherichia coli BL21-CodonPlus(DE3)-RIPL (Stratagene, USA) at 18 °C was cultured, harvested and then lysed by ultrasonication. To purify DX2, this lysate was subjected to nickel affinity chromatography followed by dialysis, tag cleavage and second nickel affinity chromatography. Size exclusion chromatography was used to further separate the oligomer fraction of AIMP2-DX2 (see reference [1] for details). During the dialysis of the elution fraction from nickel affinity chromatography to standard buffer for SUMO-tag cleavage (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM DTT) and the subsequent tag cleavage, precipitation of SUMO-DX2 was severe with loss of about 50–60% of protein. To optimize this step of purification, 500 ml of buffers with several different additives were prepared. Then, the stability of 1–2 ml of elution fractions on dialyzing to these buffers and subsequent tag cleavage was analyzed with SDS-PAGE. Osmolytes (glycerol, mannitol, betaine and trehalose), the salts of the Hofmeister series (MgSO₄, MgCl₂, Na₂SO₄, NaCl and KCl) and other common additives like EDTA, DTT and mild detergents were checked for their protective action [5], [6]. From the initial screening process, NaCl and EDTA showed the most protective effect. Both NaCl and EDTA were further screened for their protective effect at various concentrations. After several rounds of screening, the most optimal method was to mix the elution fractions from nickel affinity chromatography with EDTA at concentration of 10 mM before extensive dialysis to buffer solution 50 mM Tris–HCl pH 7.4, 1 mM DTT and 500 mM NaCl. This buffer condition was also most suitable for SUMO-tag cleavage. It should be noted that the absence of the EDTA in the buffer during tag cleavage had no effect on the stability of the protein.

These data has been successfully used to get a maximum yield (10 mg/L) of highly pure (> 95%) AIMP2-DX2 for subsequent characterization of its biophysical property in: “Purification and biophysical characterization of the AIMP2-DX2 protein” (R. Jha, H.Y. Cho, A. Ul Mushtaq, K. Lee, D.G. Kim, S. Kim, et al., 2017) [3].