Imidazole

Microcrystalline cellulose (Avicel® PH-101, ∼50 μm particle size) was purchased from Sigma-Aldrich, Co. LLC. and dried under vacuum until constant weight before use. 1-MethylImidazole, 1-butylImidazole, 1,3-propanesultone, and 1-iodooctane were purchased from Wako Pure Chemical Industries, Ltd. and purified through distillation before use. Imidazole (>98.0%), chlorosulfonic acid (>97.0%), trifluoromethanesulfonic acid (TFS, >98.0%), methanesulfonic acid (MeS, >98.0%), benzenesulfonic acid (BzS, >98.0%), glacial acetic acid (AcOH, ≥99%), and 4-nitroaniline (>99.0%) were also purchased from Wako Pure Chemical Industries, Ltd. Potassium hydroxide (KOH, >86.0%), trifluoroacetic acid (TFAc, >99.0%), trichloroacetic acid (TClAc, 99%), sulfuric acid (H₂SO₄, >96.0%), and hydrochloric acid (HCl, 35.0–37.0%) were purchased from Kanto Chemicals Co., Inc. Bis(trifluoromethanesulfonyl)imide (HTFSI) was purchased from Morita Chemical Industries, Co., Ltd.; and phosphoric acid was purchased (>99.9%) from Sigma-Aldrich, Co. LLC. Other chemicals were also commercially available and used as received unless otherwise stated.

Recombinant GST- or His₆-tagged proteins were expressed in and purified from E. coli. The BL21(DE3)pLysS bacterial cells were transformed with pGEX-6P- or pET30-based vectors, cultured, and then exposed to 0.5 mM isopropyl-β-d-thiogalactopyranoside for 16 h at 10 °C. The cells were then subjected to ultrasonic treatment, and the soluble fraction of cell lysates was purified. The expressed GST-tagged proteins were purified with glutathione-Sepharose 4B beads (Amersham Biosciences) and were eluted from the beads with reduced glutathione. The expressed His₆-tagged proteins were purified with Ni-NTA agarose (Wako) and were eluted with imidazole (Wako). Purified proteins were concentrated with an Amicon Ultra device (Merck Millipore, Tokyo, Japan) and dialyzed against PBS with a Mini Dialysis Kit (GE Healthcare).

Recombinant histidine-tagged apoAequorin (198 amino acids, Mw = 22,512.1) was expressed into the periplasmic space of E. coli cells using a piP-His-HE expression vector and purified, as previously described [19 (link)]. Coelenteramide (CTMD) was chemically synthesized according to a previously described procedure [19 (link)]. The following chemicals were obtained from commercial sources: coelenterazine (JNC. Co., Tokyo, Japan); 2-mercaptoethanol, dithiothreitol (DTT), dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid disodium salt (EDTA·2Na), and imidazole (Wako Pure Chemicals, Osaka, Japan); Butyl-Sepharose FF (GE Healthcare, Piscataway, NJ, USA). All other chemicals were of the highest commercial grade available.

We transformed E. coli strain JM109(DE3) (P9801, Promega) with the pRSET_B plasmids encoding ELP-TEMP or ELP-REF fused with an N-terminal polyhistidine tag by heat shock method at 42 °C for 45 s. We spread the transformants on an LB plate containing 100 µg/mL carbenicillin and incubated at 37 °C for overnight. We grew E. coli in a 200 mL LB medium containing 100 µg/mL carbenicillin at 23 °C for 3 days with gentle shaking at 120 rpm. We harvested the E. coli cells, suspended them in a phosphate buffered saline solution (PBS; T900, Takara Bio) containing a protease inhibitor cocktail (11873580001, Roche Diagnostics), and ultra-sonicated to lysate. We subsequently applied the proteins on the Ni–NTA agarose (30230, Qiagen) and eluted them with a TN buffer (10 mM Tris–HCl and 150 mM NaCl, pH 8.0) supplemented with 200 mM imidazole (099–00013, FUJIFILM Wako). Finally, we exchanged the solvent of the protein solution with a PBS solution (pH 7.4) by applying on a PD-10 desalting column (17085101, GE Healthcare) equilibrated with the same buffer. We concentrated the protein by ultrafiltration using a filter with a molecular weight cut-off of 50 kDa (UFC803024, Amicon Ultra-4, Merck Millipore), quickly froze the protein solution in liquid nitrogen, and stored them at − 80 °C.

Recombinant His₆-tagged proteins were expressed in and purified from Escherichia coli. The BL21(DE3)pLys bacterial cells were transformed with pET30-based vectors, cultured, and then exposed to 0.5 mM isopropyl-β-D-thiogalactopyranoside for 16 h at 10°C. The cells were then subjected to ultrasonic treatment, and the soluble fraction of the cell lysates was isolated. The expressed His₆-tagged proteins were purified with the use of Ni-NTA agarose (Wako) and were eluted with imidazole (Wako). The purified proteins were concentrated with an Amicon Ultra device (Merck Millipore) and dialyzed against PBS with the use of a Mini Dialysis Kit (GE Healthcare).

Blue-native PAGE (BN-PAGE) was performed according to Schagger (2001) (link). Mitochondrial fraction isolated from heart tissues were solubilized with solubilization buffer (1% digitonin (#044-02121, WAKO), 20 mM NaCl, 50 mM imidazole (#091-00012, WAKO) pH 7.0, 1 mM EDTA, 10% glycerol, 500 mM 6-aminocaproic acid (#103200, WAKO), protease inhibitors (#11697498001, Roche)). After centrifugation at 14,000 × g for 15 min at 4°C Coomassie Brilliant blue G-250 (0.2% final concentration) was added, and the samples were electrophoresed through 3–12% polyacrylamide gradient gels. The gels were subjected to IB using the Abs described in the figures.

The chicken CENP-C fragment (amino acids 1-302) was cloned into pET30b (Merk, 69910). The His-tag fused chicken CENP-C (1-302) protein was expressed in an Escherichia coli cell line, Rosetta2 (DE3) (Merk, C2527I). Cells expressing His-tag fused chicken CENP-C (1-302) were harvested by centrifugation, resuspended in binding buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5 mM Imidazole (Wako, 095-00015), and 1xcomplete EDTA-free proteinase inhibitor), and lysed by sonication. The lysate was clarified by centrifugation and the supernatant was incubated with Ni-NTA beads (Qiagen, 30210). After washing Ni-NTA beads with wash buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 40 mM Imidazole), bound proteins were eluted with elute buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 800 mM Imidazole). The elution was dialyzed against PBS, concentrated using Amicon-Ultra-15 10 K (Merk, UFC901008), frozen in liquid nitrogen and stored at −80 °C. The purified protein was sent to Cosmo bio to raise antibodies against chicken CENP-C in rabbits.