Hiload 16 60 superdex 200 column

Recombinant full-length UV-DDB (DDB1–DDB2 heterodimer) was expressed in Sf9 cells coinfected with recombinant baculovirus of DDB1-6xHis and DDB2-Flag, as performed previously (26 (link),27 (link)). Briefly, DDB1-6xHis and DDB2-Flag were purified using a 5 ml His-Trap HP column pre-charged with Ni²⁺ (GE Healthcare) and anti-FLAG M2 affinity gel (Sigma). The pooled anti-FLAG eluates were size fractionated on a HiLoad 16/60 Superdex 200 column (Amersham Pharmacia) in UV-DDB storage buffer (50 mM HEPES, pH 7.5, 200 mM KCl, 1 mM EDTA, 0.5 mM PMSF, 2 mM DTT, 10% glycerol and 0.02% sodium azide). Purified fractions of the DDB1–DDB2 complex from the Superdex200 were aliquoted and flash-frozen with liquid nitrogen and stored at −80°C. SMUG1 WT was purchased from NOVUS (Saint Charles, MO).

Recombinant full-length UV-DDB (DDB1-DDB2 heterodimer) was expressed in Sf9 cells coinfected with recombinant baculovirus of DDB1-His6 and DDB2-Flag, as performed previously (17 (link)). Briefly, DDB1-His6 and DDB2-Flag were purified using a 5 ml His-Trap HP column pre-charged with Ni2+ (GE Healthcare) and anti-FLAG M2 affinity gel (Sigma). The pooled anti-FLAG eluates were size fractionated on a HiLoad 16/60 Superdex 200 column (Amersham Pharmacia) in UV-DDB storage buffer (50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5, 200 mM KCl, 1 mM EDTA, 0.5 mM PMSF (phenylmethylsulfonyl fluoride), 2 mM DTT (dithiothreitol), 10% glycerol,and 0.02% sodium azide). Purified fractions of DDB1-DDB2 complex from the Superdex200 were aliquoted and flash frozen with liquid nitrogen and stored at −80°C. AAG WT was purchased from NOVUS (Saint Charles, MO) and AAG Δ80 p.E125Q (EQ) was purified as previously described (22 ).

Recombinant full-length UV-DDB (DDB1-DDB2 heterodimer) was expressed in Sf9 cells coinfected with recombinant baculovirus of His₆-DDB1 and DDB2-Flag, as performed previously (34 (link)). Briefly, a 5 ml His-Trap HP column pre-charged with Ni²⁺ (GE Healthcare) and anti-FLAG M2 affinity gel (Sigma) was used to purify DDB1-His₆ and DDB2-Flag. The pooled anti-FLAG eluate containing UV-DDB (DDB1:DDB2 at a 1:1 ratio) was purified based on size with a HiLoad 16/60 Superdex 200 column (Amersham Pharmacia) in UV-DDB storage buffer (50 mM HEPES, pH 7.5, 200 mM KCl, 1 mM EDTA, 0.5 mM PMSF, 2 mM DTT, 10% glycerol and 0.02% sodium azide). Purified fractions of DDB1–DDB2 complex from the Superdex200 were aliquoted and flash-frozen with liquid nitrogen and stored at −80°C.

A full-length AbIDH2 gene from A. baumannii was cloned into pET28b (Invitrogen, Carlsbad, CA, USA). Plasmid was then transformed into E. coli BL21 (DE3) and the recombinant protein was overexpressed. The transformed cells were grown on Luria-Bertani (LB) agar plate containing 100 μg mL⁻¹ kanamycin. A single colony was picked and grown in LB medium containing 100 μg mL⁻¹ kanamycin until OD₆₀₀ reached 0.6 at 37 °C. After induction by 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG), the cells were further grown for 20 h at 22 °C and harvested by centrifugation. E. coli BL21 cells were resuspended in lysis buffer containing 50 mM NaH₂PO₄ pH 7.5, 300 mM NaCl, and then disrupted by sonication. The recombinant AbIDH2 with 6× His-tag on its N-terminus was purified using BD TALON Metal Affinity Resin (Clontech, LaJolla, CA, USA) according to the manufacturer’s instructions. Protein was further purified by gel filtration on a Hi-Load 16/60 Superdex 200 column (Amersham Biosciences, Uppsala, Sweden). The major peak fractions were collected and concentrated for crystallization. Mutations were introduced into AbIDH2 by overlap extension, PCR-based, site-directed mutagenesis. Mutant AbIDH2 was expressed and purified as the wild-type AbIDH2.

Drosophila IntS4, IntS9, and IntS11 were co-expressed in insect cells. IntS9 and IntS11 were cloned into the pFL acceptor vector. N-terminal 6xHis-tagged IntS4 was cloned into the pSPL donor vector. These two vectors were fused by Cre recombinase. Tni insect cells (Expression Systems) (2 × 10⁶ cells·ml^–1) were infected with 16 ml of IntS4-IntS9-IntS11 P2 virus and harvested after 48 h.
For purification, the cell pellet was resuspended and lysed by sonication in 100 ml of buffer containing 20 mM Tris (pH 8.0), 250 mM NaCl, 2 mM βME, 5% (v/v) glycerol, and one tablet of protease inhibitor mixture (Sigma). The cell lysate was then centrifuged at 15,000 × g for 40 min at 4 °C. The protein complex was purified from the supernatant via nickel affinity chromatography (Qiagen). The protein complex was further purified using a Hiload 16/60 Superdex 200 column (Cytiva). The IntS4-IntS9-IntS11 complex was concentrated to 2 mg·ml^–1 in a buffer containing 20 mM Tris (pH 8.0), 300 mM NaCl, and 2 mM DTT, and stored at −80 °C.

Based on the information of DDBJ/EMBL/GenBank accession number NM_001289778.1, oligonucleotide primers (5′-ATGTCGACATGGAGCGCAGCGGTGGGAACGGCG-3′ and 5′-ATGTCGACTCAACAGAAGGTGTTCAGCGTAGTTTC-3′) were designed, and rat Map7d2 (rMap7d2) cDNA was obtained by PCR using rat cDNA as a template. Expression vectors for rMap7d2 were constructed in pCMV5-Myc (Nakanishi et al, 1997 (link)), pQE9 (QIAGEN), pGEX-5X-3 (Cytiva), pcDNA3.1/V5-His (Thermo Fisher Scientific), pCLXSN-GFP (Reiley et al, 2005 (link)), and pEGFP-N3 (Clontech). GST-fused proteins were expressed in Escherichia coli and purified using glutathione-Sepharose beads (Cytiva), respectively. GST-rMap7D2 (full length) was further purified by gel filtration using a HiLoad 16/60 Superdex 200 column (Cytiva).