The strains and plasmids used in this study were listed in additional file (Additional file 1 : Table S1). E. coli Trans5α (TransGen, Beijing, China) was used for plasmid construction. E. coli BL21(DE3) and E. coli BL21(DE3)pLysS (TransGen, Beijing, China) were used as the hosts for Bgl1A(A24S/F297Y) production. Ampicillin, chloramphenicol, and IPTG were purchased from Sangon Biotech (Shanghai, China). p-Nitrophenyl β-D-glucopyranoside (pNPG) was from Sigma-Aldrich (St. Louis, MO, USA). The insoluble microcrystalline cellulose with an average particle size of 25 μm was acquired from Aladdin Chemistry (Shanghai, China). Ni2+-charged chelating sepharose fast flow was purchased from GE Healthcare (Uppsala, Sweden). All other chemicals were of analytical grade unless otherwise specified. Standard TB medium (per liter contains 4 g glycerol, 24 g yeast extract, 12 g peptone, 17 mM KH2PO4, and 72 mM K2HPO4) was used as culture medium in 250-mL Erlenmeyer flasks. Modified TB medium (per liter contains 15 g glycerol) was employed in high cell density culture (HCDC), the feeding solutions contained 45 g tryptone, 45 g yeast extract, and 500 g glycerol per liter.
Ni2 charged chelating sepharose fast flow
Manufactured by GE Healthcare
Sourced in Sweden
Ni2+ charged Chelating Sepharose Fast Flow is a chromatography resin designed for the purification of histidine-tagged recombinant proteins. The resin utilizes Ni2+ ions immobilized on a Sepharose matrix to provide a specific and reversible interaction with the histidine-rich protein tag, allowing for selective capture and purification.
Automatically generated - may contain errors
Lab products found in correlation
Superdex 75 column, by GE Healthcare (2 mentions)
P nitrophenyl β d glucopyranoside pnpg, by Merck Group (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Sangon (1 mentions)
E coli trans5α, by Transgene (1 mentions)
E coli bl21 de3 plyss, by Transgene (1 mentions)
Ampicillin, by Sangon (1 mentions)
Chloramphenicol, by Sangon (1 mentions)
Pdest17, by Thermo Fisher Scientific (1 mentions)
6 protocols using ni2 charged chelating sepharose fast flow
1
Bgl1A(A24S/F297Y) production protocol
2
Cellulose-Bound Bgl1A(A24S/F297Y) Purification
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The cell lysate was centrifuged at 12,000×g for 10 min. The supernatant was withdrawn and diluted to 10 U/mL of β-glucosidase activity, the pH of crude enzyme solution was adjusted to 6.5 and NaCl was supplemented at a final concentration of 200 mM. Finally, 0.5 g cellulose was added into 10 mL of crude enzyme solution. The mixture was incubated at 25 °C under mild shaking for 1 h, followed by centrifugation at 4000×g for 5 min. The cellulose bound Bgl1A(A24S/F297Y)-CBM was in sediment and was washed 3 times with Na2HPO4-citric acid buffer (50 mM, pH 6.5) to remove the unbound or loosely bound protein. The protein binding efficiency was calculated by measuring the β-glucosidase activity in supernatant and cellulose suspension. Bgl1A(A24S/F297Y) with C-terminal His6 purified by Ni2+ charged Chelating Sepharose Fast Flow (GE Healthcare, Uppsala, Sweden) was used as control.
3
Cloning and Purification of KmGLK1 and KmHXK1
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The NdeI site in KmGLK1 was removed by silent mutation. Using pKmGLK-T as template, a C- terminal fragment of KmGLK1 was first amplified by GLKdNdeI-F and KmGLK-XhoI-R (Supplementary Table S1 ) to eliminate the NdeI site. And the N-terminal fragment of KmGLK1 was amplified by KmGLK-NdeI-F and GLKdNdeI-R (Supplementary Table S1 ). Then the whole KmGLK1 with silent mutation was amplified with KmGLK-NdeI-F and KmGLK-XhoI-R through overlap PCR with above two fragments. The KmHXK1 gene was amplified from pKmHXK-T with primer pair KmHXK-NdeI-F and KmHXK-XhoI-R. Amplified KmGLK1 and KmHXK1 were digested by NdeI and XhoI and inserted into pET22b vector with a His-tag fused to the protein C-terminal. Resultant plasmids were named as pET22b-KmGLK and pET22b-KmHXK (Table 3 ), respectively. Then the plasmids were transformed into E. coli BL21(DE3) to express recombinant enzymes. The expression was induced with 1.0 mM IPTG in LB medium at 37 °C for 4 h when OD600 reach 0.5~0.8. After E. coli cells were recovered through centrifugation and lysed by sonication, the recombinant enzymes in supernatant of lysed E. coli cell were purified by Ni2+ charged Chelating Sepharose Fast Flow (GE Healthcare, Uppsala, Sweden) according the product instruction.
4
Nin Protein Purification and Antibody Generation
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Sequences corresponding to amino acids 829–1026 of Nin-PB were cloned into pDEST-17 (Invitrogen) for expression of a 6×His-tagged protein in E. coli strain BL21(DE3)pLysE. Nin protein was isolated from E. coli in inclusion bodies, dissolved in 6M guanidine hydrochloride, centrifuged at 12,000 × g for 20 min, and purified by immobilized metal affinity chromatography with Ni2+-charged Chelating Sepharose Fast Flow (GE Healthcare, Little Chalfont, United Kingdom). Purified protein was dialyzed against phosphate-buffered saline (PBS), and 3 mg purified by preparative minigel using 10% SDS–PAGE. The protein band was excised from the gel, and antibodies were raised in rabbits by Cocalico Biologicals. This antibody was used for Western blotting. A second antibody, used for immunostaining, was generated by immunizing rabbits (Covance, Princeton, NJ) with a synthetic peptide corresponding to amino acids 1074–1091 of Nin-PB. Antibodies were affinity-purified by peptide affinity chromatography using sulfolink resin (Pierce, Thermo Fisher).
5
Expression and Purification of Dhh1 Variants
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Expression and purification of mCherry-tagged Dhh1, Dhh148–425, Dhh1DQAD, and non-tagged MIF4G-Not1 was performed as previously described12 . Briefly, competent Escherichia coli BL21-Gold (DE3) strains were transformed via heat shock at 42 °C with plasmids carrying the genes for Dhh1 (pKW3631), Dhh148–425 (pKW4063), Dhh1DQAD (pKW3632) or Not1MIF4G (pKW3469). Each plasmid was carrying sequences for a 6× His tag and ampicillin resistance. Cells were cultured in LB medium at 37 °C and protein expression was induced by the addition of 0.5 mM (0.2 mM for Not1MIF4G) isopropyl-beta-D-1-thiogalactopyranoside. After harvesting, cells were resuspended in lysis buffer (pH 7.5, 300 mM NaCl, 50 mM Tris, 10 mM imidazole, 10% glycerol) and lysed by sonication. Protein purification was performed via affinity chromatography using Ni2+ charged Fast Flow Chelating Sepharose (GE Healthcare) as a stationary phase. This step was followed by size exclusion chromatography on a Superdex 75 column (GE Healthcare) using a solution at pH 7.5, 300 mM NaCl, 25 mM Tris, 2 mM 2-Mercaptoethanol, and 10% glycerol as elution buffer. Purified fractions were pooled, concentrated, and flash-frozen in liquid nitrogen. The phase diagram of Dhh1 was typically analyzed in 30 mM HEPES-KOH buffer at pH 7.4 supplied with 150 mM KCl and 2 mM MgCl2.
6
Expression and Purification of mCherry-tagged Dhh1
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Expression and purification of mCherry-tagged Dhh1 (mCh-Dhh1), Dhh1 48-425 , Dhh1 DQAD and non-tagged MIF4G-Not1 was performed as previously described (Mugler et al., 2016) . Briefly, competent Escherichia coli BL21-Gold (DE3) strains were transformed via heat shock at 42°C with plasmids carrying the genes for Dhh1 (pKW3631), Dhh1 48-425 (pKW4063), Dhh1 DQAD (pKW3632) or Not1 MIF4G (pKW3469). Each plasmid was carrying sequences for a 6x His tag and ampicillin resistance. Cells were cultured in LB medium at 37 °C and protein expression was induced by addition of 0.5 mM (0.2 mM for Not1 MIF4G ) isopropyl-beta-D-1thiogalactopyranoside (IPTG). After harvesting, cells were resuspended in lysis buffer (pH 7.5, 300 mM NaCl, 50 mM Tris, 10 mM imidazole, 10 % glycerol) and lysed by sonication. Protein purification was performed via affinity chromatography using Ni 2+ charged Fast Flow Chelating Sepharose (GE Healthcare) as stationary phase. This step was followed by size exclusion chromatography on a Superdex 75 column (GE Healthcare) using a solution at pH 7.5, 300 mM NaCl, 25 mM Tris, 2 mM 2-Mercaptoethanol and 10 % glycerol as elution buffer.
Purified fractions were pooled, concentrated and flash-frozen in liquid nitrogen. The phase diagram of Dhh1 was typically analyzed in buffer b-150, a 30 mM HEPES-KOH buffer at pH 7.4 supplied with 150 mM KCl and 2 mM MgCl2.
Purified fractions were pooled, concentrated and flash-frozen in liquid nitrogen. The phase diagram of Dhh1 was typically analyzed in buffer b-150, a 30 mM HEPES-KOH buffer at pH 7.4 supplied with 150 mM KCl and 2 mM MgCl2.
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