Isopropyl β d 1 thiogalactopyranoside (iptg)

Ran cDNA was amplified by PCR using primers (5′-AAT​CAT​ATG​GCC​GCC​CAG​GGA​GAG​C-3′ and 5′-AAT​GAA​TTC​TCA​CAG​GTC​ATC​ATC​CTC​ATC​TGG​GAG​AG-3′) and cloned into the pET28b vector using NdeI and EcoRI. The T24N mutant was generated using a KOD Plus Mutagenesis Kit (Toyobo) with primers (5′-GCA​CCG​GGA​AGA​ATA​CCT​TCG​TGA​AGC​GCC​ACT​TGA​CGG​GCG​AG-3′ and 5′-GCT​TCA​CGA​AGG​TAT​TCT​TCC​CGG​TGC​CGC​CGT​CGC​CCA​CCA​GG-3′). pET28b-RanWT or pET28b-RanT24N plasmids were transformed into BL21(DE3). His-Ran recombinant proteins were expressed with 1 mM IPTG (Nacalai) at 28°C for 2 h and purified by the Qiagen Ni-NTA protocol for native conditions (Qiagen; lysis buffer: 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0; wash buffer: 50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, pH 8.0; and elution buffer: 50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0). The buffer of the eluted fraction was changed to PBS using a PD-10 column (GE), and His-Ran proteins were concentrated using a Vivaspin 500 (GE).

The plasmids encoding each ribosomal protein were purified using the ASKA library [36 (link)]. Then, the plasmids were introduced into E. coli BL21 (DE3) lacZ::kmr. The cells were grown at 37 °C to OD₆₀₀ = 0.5–0.6 in 5 mL LB medium containing 100 μg/ml ampicillin (Viccillin^® for injection, Meiji Seika Pharma), incubated at 37 °C for 3 h with 0.1 or 1 mM IPTG (Nacalai tesque), and then centrifuged at 3,000 rpm for 5 min. Then, the pellets of the cells were suspended in 200 μL of lysis buffer of 2 M urea, 50 mM ammonium bicarbonate, and with a pH of 8.0 and transferred into 1.5 mL microtubes in an ice-water bath. The cells were disrupted using a sonicator (BIORUPTOR UCD-250, Cosmo Bio, Tokyo, Japan) for 10 min at 4 °C (Level 5, a 30-s sonication with 30-s interval). Then, the lysates were centrifuged at 14,000 g for 10 min at 4 °C, and the supernatants were collected. The ribosomal proteins were quantified using the LCMS-8060 (Shimadzu, Kyoto, Japan) as described below.

E. coli XL10-Gold {Tet^rΔ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F' proAB lacI^qZΔM15 Tn10 (Tet^r) Amy Cam^r]} (Stratagene. La Jolla, CA) was used for cloning, while XL1-Blue {recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F' proAB lacI^qZΔM15 Tn10 (Tet^r)]} (Stratagene) was used for carotenoid production experiments. Cells were grown Luria-Bertani (LB) in Lennox media for cloning or preculture and in Terrific Broth (TB) media for carotenoid production. Carbenicillin (50 μg ml⁻¹; Sigma-Aldrich, St Louis, MO) and/or chloramphenicol (30 μg ml⁻¹; Nacalai Tesque, Kyoto, Japan) were supplemented where appropriate. For protein overexpression and purification, BL21-AI (Life Technologies, Carlsbad, CA) was used. For induction, 0.2% (w/v) L-arabinose (Nacalai Tesque) and 0.1 mM IPTG (Nacalai Tesque) was added as an inducer.

We obtained antibiotics and reagents used in this study except ampicillin, kanamycin, chloramphenicol and isopropyl β-D-1-thiogalactopyranoside (IPTG) from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). ampicillin, kanamycin, chloramphenicol and IPTG were purchased from Nacalai tesque (Kyoto, Japan).

The MB2 region of MYCL-WT or -W96E was cloned into pGEX-6P-1. The plasmids were transformed into BL21 E. coli (DE3) (L1198, Promega) competent cells. The fusion proteins, GST-MYCL-WT-MB2 and GST-MYCL-W96E-MB2, were induced by treatment with 0.5 mM IPTG (19742-94, Nacalai Tesque) for 4 h at 37 °C. The proteins were purified using glutathione Sepharose beads (17-0756-01, GE Healthcare). Human iPSCs or reprogramming HDFs were lysed in RIPA buffer (20 mM Tris/HCl (pH 7.6) (35436-01, Nacalai Tesque), 1% NP-40 (25223-75, Nacalai Tesque), 0.1% SDS, 150 mM NaCl (31320-05, Nacalai Tesque), and protease inhibitor (25955-11, Nacalai Tesque)) and then centrifuged. Cell lysates (supernatant) were transferred into a column (29922, Thermo Fisher Scientific) packed with beads conjugated with GST- MYCL-WT or -W96E proteins. After washing, binding proteins were eluted in lysis buffer (12 mM sodium deoxycholate (190-08313, Wako), 12 mM sodium lauroyl sarcosinate (192-10382, Wako), and 100 mM Tris-HCl (pH9.0) (314-90381, NIPPON GENE)) for the MS analysis. The iPSC lysates were prepared 6 days after passaging in two 10-cm dishes (n = 1), and reprogramming HDF lysates were prepared 3 days after SeV transduction in five 10-cm dishes (n = 1).