Isopropyl β d thiogalactopyranoside iptg

Recombinant proteins were purified as previously described [17 (link)]. GST‐E6AP, His6‐MCM6, and their respective truncation proteins and His6‐MCM2/3/4/5/7 were expressed in BL21 E. coli cells. After isopropyl‐β‐d‐thiogalactopyranoside (IPTG, Sangon, China) induction, bacterial cells were pelleted, lysed in PBS buffer and incubated with glutathione or Ni2 + TA beads (Sangon, China) to enrich the respective proteins, followed by elution with 300 mm imidazole (Sangon) or 20 mm L‐glutathione reduced (Sangon) dissolved in PBS buffer, and then dialysis in PBS buffer supplemented with 20% glycerol before being aliquoted and preserved at −80 °C.

The bacterial strain and plasmids involved in this study are listed in Table 1. E. coli TOP10 was used as the host, and cultured in M9 medium (0.68% Na₂HPO₄, 0.3% KH₂PO₄, 0.05% NaCl, 0.1% NH₄Cl, 0.01% MgSO₄, and 0.001% CaCl₂) supplemented with glucose at 0.4%, IPTG at 0.1 mM, and ampicillin at 50 μg/mL. Stock solutions of CaCl₂, MgCl₂, FeSO₄, MnSO₄, NiSO₄, CuSO₄, ZnSO₄, CdCl₂, Pb(NO₃)₂, and HgCl₂ were freshly prepared with analytical grade chemicals and distilled water. SP sepharose fast flow was obtained from GE Healthcare (USA). Isopropyl-β-D-thiogalactopyranoside (IPTG) and o-nitrophenyl-β-D-galactopyranoside (ONPG) were obtained from Sangon Biotech (Shanghai, China). All oligonucleotide primers and DNA fragments were synthesized by Sangon Biotech (Shanghai, China).

The ORF41 gene was amplified from the DNA of the purified phage SH-KP156570 using primers 6570-ORF41-F (5′-CAGCAGCAGACGGGAGGATCCATGTCCACGATTACACA ATTC-3′) and 6570-ORF41-R (5′-CTCGAGTGCGGCCG CAAGCTTTTAGTTACTTCTCTCTTCAGC-3′). The 2292-base PCR amplification product was cloned into the N-terminal 6 × His labeled pSUMO3 expression vector (LifeSensors, United States) via BamHI and HindIII sites (New England Biolabs). The recombinant plasmid was verified by DNA sequencing and transformed into E. coli BL21 (DE3). The BL21 cells were cultivated to OD₆₀₀ = 0.8. Then, the BL21 cells were induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Sangon Biotech, China). The mixture was centrifuged and the supernatant was discarded. The sediment was resuspended with lysis buffer [20 mM Tris–HCl (pH = 8.0), 0.5M NaCl, 10% glycerol] and phenylmethylsulfonyl fluoride (PMSF, Sangon Biotech, China) to a final concentration of 0.1 mg/mL. After centrifuged, the supernatant was collected. The Ni-NTA column (GE Healthcare, United States) was prerinsed using lysis buffer. After protein binding to Ni-NTA column, it was eluted with a gradient of 20 and 40 mM imidazole, and finally eluted with 300 mM imidazole. Then digested by SUMO protease (LifeSensors, United States). The purified depolymerase was confirmed by SDS-PAGE electrophoresis and named K19-Dpo41.

E. coli strain BL21 (DE3) was used as the host for protein expression. The lipids POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), NBD-PE (1-oleoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] dodecanoyl}-sn-glycero-3-phosphoethanolamine), cardiolipin, and CHAPS were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Horse cytochrome c was purchased from Sigma (Merck KGaA, Darmstadt, Germany). Isopropyl-β-D-thiogalactopyranoside (IPTG) and all the other chemicals were from Sangon Biotech (Shanghai, China).

The AT-ATA cDNA sequence, including the NcoI and XhoI restriction sites, and all the primers used for mutagenesis were synthesized by General Biosystems (AnHui) Co., Ltd. (Chuzhou, China). The recombinant plasmid pET28a-AT-ATA was used as the template. PrimeSTAR^® Max DNA polymerase was acquired from Takara Biotechnology (Dalian, China). Lactate dehydrogenase (LDH), dimethyl sulfoxide (DMSO), l-alanine, acetophenone, 2-butylamine, imidazole, isopropyl-β-d-thiogalactopyranoside (IPTG), and Ni-IDA-Sefinose™ resin kits were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The SYPRO orange dye was purchased from Invitrogen (Carlsbad, CA, USA).