Expression and purification of MinD and its mutant

In this study, all Escherichia coli cells were cultivated in LB medium. To construct pET15b-MinD, the MinD gene was cloned from the E. coli MG1655 genome by PCR into pET15b (Merck Millipore, Billerica, MA, USA) by Gibson assembly (New England Biolabs, Ipswich, MA, USA). To construct pET15-sfGFP-minD, the sfGFP gene was amplified from pET29-sfGFP (Fujiwara and Doi, 2016) by PCR and cloned into MinD gene by Gibson assembly. It is transcribed to insert amino acids into the N-terminal of MinD. To construct pET15-sfGFP-MinD^D40AΔ10, the D40A mutation, which results in the adeletion of the C-terminal 10 amino acids of the MinD protein, were introduced into pET15-sfGFP-minD by using the PrimeSTAR Max mutagenesis protocol (TaKaRa, Shiga, Japan). Similarly, the K11A mutation was introduced into the pET15-sfGFP-minD construct in order to produce pET15-sfGFP-MinD^K11A. E. coli BL21-CodonPlus(DE3)-RIPL (Agilent Technologies, Santa Clara, CA, USA) cells were transformed with the resultant plasmids.

Protein expression was induced by 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at OD₆₀₀ = 0.1–0.2 and further cultivation at 37°C for 3 to 4 hr. The cells were collected by centrifugation and suspended in LS buffer [50 mM NaH₂PO₄ (pH 7.6), 300 mM NaCl, 10 mM imidazole, 10 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMSF)] with 0.2 mM ADP-Mg. The collected cells were disrupted by sonication using a Sonifier250 (Branson, Danbury, CT, USA), and the supernatant of the crude extract was fractionated by centrifugation at 20,000 g at 4°C for 30 min. To purify His-tagged proteins, the crude extracts mixed with cOmplete His-Tag purification resin (Roche, Basel, Switzerland) were loaded onto a polyprep chromatography column (Bio-Rad, Hercules, CA, USA), and washed with 25 mL WS buffer [50 mM NaH₂PO₄ (pH 7.6), 300 mM NaCl, 20 mM imidazole, 10% glycerol, 0.1 mM EDTA, and 0.1 mM PMSF]. His-tagged proteins were eluted with EL buffer [50 mM NaH₂PO₄ (pH 7.6), 300 mM NaCl, 250 mM imidazole, 10% glycerol, 0.1 mM EDTA, and 0.1 mM PMSF]. EL buffer was exchanged with storage buffer [50 mM HEPES-KOH (pH 7.6), 150 mM GluK, 10% glycerol, 0.1 mM EDTA] with 0.2 mM ADP-Mg by ultrafiltration using AmiconUltra-15 10 k and AmiconUltra-0.5 30 k filters (Merck Millipore).

For pull-down assay, His-sfGFP-MinD^D40AΔ10 was treated with thrombin (Wako, Osaka Japan) in the storage buffer at 4°C overnight. Then, the cleaved His-Tag (2 kDa) was removed from the sfGFP-MinD^D40AΔ10 (55 kDa) solution by ultrafiltration using AmiconUltra-0.5 50 k filters (Merck Millipore). Proteins in the storage buffer were stored at −80°C. Protein purity and concentrations were estimated by Comassie Brilliant Blue (CBB) staining after separating by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and bicinchoninic acid (BCA) assay.