Escherichia coli bl21 de3

The S2-ALDH (1365 bp) gene (Supplementary Fig. S1) identified in the Flavobacterium PL002 genome sequence^{42 (link)} was synthesized (ATG Biosynthetics GmbH, Merzhausen, Germany) and cloned into the pHAT2 His-tag expression vector (EMBL, Heidelberg, Germany) using the NcoI/BamHI restriction sites. Gene expression of the resulting pS2-ALDH recombinant plasmid was performed in Escherichia coli BL21(DE3) (Thermo Fisher Scientific, Massachusetts, USA) after induction for 6 h at 25 °C in the presence of 1 mM IPTG. The induced cells were separated by centrifugation at 9000 × g for 10 min (4 °C) and stored at − 80 °C.

The human Myo1g IQ-Tail was described previously [8 (link)]. Briefly, the construct comprising aa 702–1018 was subcloned into pDes17 by Gateway cloning (Invitrogen, Carlsbad, CA, USA), which generates 6x His-tagged N-terminal proteins. The plasmids were transformed into Escherichia coli BL21DE3 (Thermo Scientific, Waltham, MA, USA) competent cells, and protein expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Promega, Madison, WI, USA) for 5 h at 37 °C. Cells were lysed by sonication in lysis buffer (300 mM NaCl, 50 mM NaH₂PO₄, 10 mM imidazole, 6 M urea, pH 8, and a protease inhibitor cocktail-EDTA free (Thermo Scientific, Rockford, IL, USA) on ice. After centrifugation, cell lysates were purified by the batch method using 6% Cobalt-IDA Agarose (Jena Bioscience, Jena, Germany) under constant agitation at 4 °C overnight, followed by 4 washes (300 mM NaCl, 50 mM NaH₂PO₄, 10 mM imidazole, 6 M urea, pH 8). Bound proteins were eluted with elution buffer (300 mM NaCl, 50 mM NaH₂PO₄, 350 mM imidazole, 6 M urea, pH 8), and the filtrate was concentrated with Amicon Ultra-15 (Merck Millipore, Cork, Ireland) through a 0.22 µm mesh, and analyzed by SDS-PAGE and Western blot.

The mutated DEK sequences (DEK SUMOmut 61-62, 144, 145 or 261) were inserted into a GST expression plasmid (pGEX 4T-1), transformed into Escherichia coli BL21(DE3) (Thermo Fisher Scientific) and purified as described previously (Ganz et al., 2019 (link)). Briefly, protein expression was induced with 0.5 mM IPTG (Sigma-Aldrich) for 1.5 h. Bacteria were harvested via centrifugation at 4600 g for 15 min at 4°C and resuspended in resuspension buffer (20 mM Tris-HCl pH 8.0, 1 M NaCl, 0.5 mM EDTA and 1 mM DTT) and frozen in liquid nitrogen. After thawing, bacteria were sonicated on ice and 0.5% NP-40 (Sigma-Aldrich) was added and centrifuged at 18,000 g for 30 min at 4°C. The supernatant was added to 200 µl Glutathione-Sepharose 4B-beads (GE Healthcare) and incubated for 2 h at 4°C. After centrifugation, beads were washed with wash buffer with decreasing NaCl concentrations (500 mM, 300 mM and 20 mM) and GST-tagged DEK was eluted with 200 µl elution buffer [200 mM Tris-HCl pH 8.0, 200 mM NaCl, 40 mM reduced glutathione (Sigma-Aldrich) and 10% glycerol] for 1 h at 4°C. The supernatant was frozen in liquid nitrogen and stored at −80°C.